Chiral design

ABSTRACT

The present invention relates to chirally controlled oligonucleotides of select designs, chirally controlled oligonucleotide compositions, and methods of making and using the same. In some embodiments, a provided chirally controlled oligonucleotide composition provides different cleavage patterns of a nucleic acid polymer than a reference oligonucleotide composition. In some embodiments, a provided chirally controlled oligonucleotide composition provides single site cleavage within a complementary sequence of a nucleic acid polymer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry of PCT/IB2015/000395, filedJan. 15, 2015, which claims priority to U.S. Provisional ApplicationSer. No. 61/928,405, filed Jan. 16, 2014, and 62/063,359, filed Oct. 13,2014, the entirety of each of which is incorporated herein by reference.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 26, 2015, isnamed SequenceListing.txt and is 189,368 bytes in size.

BACKGROUND OF THE INVENTION

Oligonucleotides are useful in therapeutic, diagnostic, research andnanomaterials applications. The use of naturally occurring nucleic acids(e.g., unmodified DNA or RNA) for therapeutics can be limited, forexample, because of their instability against extra- and intracellularnucleases and/or their poor cell penetration and distribution.Additionally, in vitro studies have shown that properties of antisenseoligonucleotides such as binding affinity, sequence specific binding tothe complementary RNA (Cosstick and Eckstein, 1985; LaPlanche et al.,1986; Latimer et al., 1989; Hacia et al., 1994; Mesmaeker et al., 1995),and stability to nucleases can be affected by the absolutestereochemical configurations of the phosphorus atoms (Cook, et al.US005599797A). Therefore, there is a need for new and improvedoligonucleotides and oligonucleotide compositions, such as, e.g., newantisense and siRNA oligonucleotides and oligonucleotide compositions.

SUMMARY OF THE INVENTION

Among other things, the present invention encompasses the recognitionthat stereorandom oligonucleotide preparations contain a plurality ofdistinct chemical entities that differ from one another in thestereochemical structure of individual backbone chiral centers withinthe oligonucleotide chain. Moreover, the present invention encompassesthe insight that it is typically unlikely that a stereorandomoligonucleotide preparation will include every possible stereoisomer ofthe relevant oligonucleotide. Thus, among other things, the presentinvention provides new chemical entities that are particularstereoisomers of oligonucleotides of interest. That is, the presentinvention provides substantially pure preparations of singleoligonucleotide compounds, where a particular oligonucleotide compoundmay be defined by its base sequence, its length, its pattern of backbonelinkages, and its pattern of backbone chiral centers.

The present invention demonstrates, among other things, that individualstereoisomers of a particular oligonucleotide can show differentstability and/or activity from each other. Moreover, the presentdisclosure demonstrates that stability improvements achieved throughinclusion and/or location of particular chiral structures within anoligonucleotide can be comparable to, or even better than those achievedthrough use of certain modified backbone linkages, bases, and/or sugars(e.g., through use of certain types of modified phophates,2′-modifications, base modifications, etc.).

Among other things, the present invention recognizes that properties andactivities of an oligonucleotide can be adjusted by optimizing itspattern of backbone chiral centers. In some embodiments, the presentinvention provides compositions of oligonucleotides, wherein theoligonucleotides have a common pattern of backbone chiral centers which,unexpectedly, greatly enhances the stability and/or biological activityof the oligonucleotides. In some embodiments, a pattern of backbonechiral centers provides increased stability. In some embodiments, apattern of backbone chiral centers provides surprisingly increasedactivity. In some embodiments, a pattern of backbone chiral centersprovides increased stability and activity. In some embodiments, when anoligonucleotide is utilized to cleave a nucleic acid polymer, a patternof backbone chiral centers of the oligonucleotide, surprisingly byitself, changes the cleavage pattern of a target nucleic acid polymer.In some embodiments, a pattern of backbone chiral centers effectivelyprevents cleavage at secondary sites. In some embodiments, a pattern ofbackbone chiral centers creates new cleavage sites. In some embodiments,a pattern of backbone chiral centers minimizes the number of cleavagesites. In some embodiments, a pattern of backbone chiral centersminimizes the number of cleavage sites so that a target nucleic acidpolymer is cleaved at only one site within the sequence of the targetnucleic acid polymer that is complementary to the oligonucleotide. Insome embodiments, a pattern of backbone chiral centers enhances cleavageefficiency at a cleavage site. In some embodiments, a pattern ofbackbone chiral centers of the oligonucleotide improves cleavage of atarget nucleic acid polymer. In some embodiments, a pattern of backbonechiral centers increases selectivity. In some embodiments, a pattern ofbackbone chiral centers minimizes off-target effect. In someembodiments, a pattern of backbone chiral centers increase selectivity,e.g., cleavage selectivity between two target sequences differing onlyby a single nucleotide polymorphism (SNP).

All publications and patent documents cited in this application areincorporated herein by reference in their entirety.

DEFINITIONS

Aliphatic: The term “aliphatic” or “aliphatic group”, as used herein,means a straight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic or polycyclic hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic (also referred to herein as “carbocycle” “cycloaliphatic” or“cycloalkyl”), that has a single point of attachment to the rest of themolecule. In some embodiments, aliphatic groups contain 1-50 aliphaticcarbon atoms. Unless otherwise specified, aliphatic groups contain 1-10aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-6 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-5 aliphatic carbon atoms. In other embodiments, aliphaticgroups contain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic or bicyclic C₃-C₁₀ hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic, that has a single point of attachment to the rest ofthe molecule. In some embodiments, “cycloaliphatic” (or “carbocycle” or“cycloalkyl”) refers to a monocyclic C₃-C₆ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkylene: The term “alkylene” refers to a bivalent alkyl group. An“alkylene chain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein nis a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3,from 1 to 2, or from 2 to 3. A substituted alkylene chain is apolymethylene group in which one or more methylene hydrogen atoms arereplaced with a substituent. Suitable substituents include thosedescribed below for a substituted aliphatic group.

Alkenylene: The term “alkenylene” refers to a bivalent alkenyl group. Asubstituted alkenylene chain is a polymethylene group containing atleast one double bond in which one or more hydrogen atoms are replacedwith a substituent. Suitable substituents include those described belowfor a substituted aliphatic group.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, a genetically-engineered animal,and/or a clone.

Approximately: As used herein, the terms “approximately” or “about” inreference to a number are generally taken to include numbers that fallwithin a range of 5%, 10%, 15%, or 20% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evidentfrom the context (except where such number would be less than 0% orexceed 100% of a possible value). In some embodiments, use of the term“about” in reference to dosages means ±5 mg/kg/day.

Aryl: The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”may be used interchangeably with the term “aryl ring.” In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl,” as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, 10, 15, 20 or more amino acids are requiredto be characteristic of a protein. In general, a characteristic portionis one that, in addition to the sequence identity specified above,shares at least one functional characteristic with the relevant intactprotein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Characteristic structural element: The term “characteristic structuralelement” refers to a distinctive structural element (e.g., corestructure, collection of pendant moieties, sequence element, etc) thatis found in all members of a family of polypeptides, small molecules, ornucleic acids, and therefore can be used by those of ordinary skill inthe art to define members of the family.

Comparable: The term “comparable” is used herein to describe two (ormore) sets of conditions or circumstances that are sufficiently similarto one another to permit comparison of results obtained or phenomenaobserved. In some embodiments, comparable sets of conditions orcircumstances are characterized by a plurality of substantiallyidentical features and one or a small number of varied features. Thoseof ordinary skill in the art will appreciate that sets of conditions arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder the different sets of conditions or circumstances are caused by orindicative of the variation in those features that are varied.

Dosing regimen: As used herein, a “dosing regimen” or “therapeuticregimen” refers to a set of unit doses (typically more than one) thatare administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, all doses within a dosing regimen are of thesame unit dose amount. In some embodiments, different doses within adosing regimen are of different amounts. In some embodiments, a dosingregimen comprises a first dose in a first dose amount, followed by oneor more additional doses in a second dose amount different from thefirst dose amount. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount same as the first dose amount.

Equivalent agents: Those of ordinary skill in the art, reading thepresent disclosure, will appreciate that the scope of useful agents inthe context of the present invention is not limited to thosespecifically mentioned or exemplified herein. In particular, thoseskilled in the art will recognize that active agents typically have astructure that consists of a core and attached pendant moieties, andfurthermore will appreciate that simple variations of such core and/orpendant moieties may not significantly alter activity of the agent. Forexample, in some embodiments, substitution of one or more pendantmoieties with groups of comparable three-dimensional structure and/orchemical reactivity characteristics may generate a substituted compoundor portion equivalent to a parent reference compound or portion. In someembodiments, addition or removal of one or more pendant moieties maygenerate a substituted compound equivalent to a parent referencecompound. In some embodiments, alteration of core structure, for exampleby addition or removal of a small number of bonds (typically not morethan 5, 4, 3, 2, or 1 bonds, and often only a single bond) may generatea substituted compound equivalent to a parent reference compound. Inmany embodiments, equivalent compounds may be prepared by methodsillustrated in general reaction schemes as, for example, describedbelow, or by modifications thereof, using readily available startingmaterials, reagents and conventional or provided synthesis procedures.In these reactions, it is also possible to make use of variants, whichare in themselves known, but are not mentioned here.

Equivalent Dosage: The term “equivalent dosage” is used herein tocompare dosages of different pharmaceutically active agents that effectthe same biological result. Dosages of two different agents areconsidered to be “equivalent” to one another in accordance with thepresent invention if they achieve a comparable level or extent of thebiological result. In some embodiments, equivalent dosages of differentpharmaceutical agents for use in accordance with the present inventionare determined using in vitro and/or in vivo assays as described herein.In some embodiments, one or more lysosomal activating agents for use inaccordance with the present invention is utilized at a dose equivalentto a dose of a reference lysosomal activating agent; in some suchembodiments, the reference lysosomal activating agent for such purposeis selected from the group consisting of small molecule allostericactivators (e.g., pyrazolpyrimidines), imminosugars (e.g., isofagomine),antioxidants (e.g., n-acetyl-cysteine), and regulators of cellulartrafficking (e.g., Rab 1a polypeptide).

Heteroaliphatic: The term “heteroaliphatic” refers to an aliphatic groupwherein one or more units selected from C, CH, CH₂, or CH₃ areindependently replaced by a heteroatom. In some embodiments, aheteroaliphatic group is heteroalkyl. In some embodiments, aheteroaliphatic group is heteroalkenyl.

Heteroaryl: The terms “heteroaryl” and “heteroar-,” used alone or aspart of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,”refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ringatoms; having 6, 10, or 14 π electrons shared in a cyclic array; andhaving, in addition to carbon atoms, from one to five heteroatoms. Theterm “heteroatom” refers to nitrogen, oxygen, or sulfur, and includesany oxidized form of nitrogen or sulfur, and any quaternized form of abasic nitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-,” as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

Heteroatom: The term “heteroatom” means one or more of oxygen, sulfur,nitrogen, phosphorus, boron, selenium, or silicon (including, anyoxidized form of nitrogen, boron, selenium, sulfur, phosphorus, orsilicon; the quaternized form of any basic nitrogen or; a substitutablenitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl)).

Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,”“heterocyclic radical,” and “heterocyclic ring” are used interchangeablyand refer to a stable 3- to 7-membered monocyclic or 7-10-memberedbicyclic heterocyclic moiety that is either saturated or partiallyunsaturated, and having, in addition to carbon atoms, one or more,preferably one to four, heteroatoms, as defined above. When used inreference to a ring atom of a heterocycle, the term “nitrogen” includesa substituted nitrogen. As an example, in a saturated or partiallyunsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl), or ⁺N (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Intraperitoneal: The phrases “intraperitoneal administration” and“administered intraperitonealy” as used herein have their art-understoodmeaning referring to administration of a compound or composition intothe peritoneum of a subject.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.,animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, and/or microbe).

Lower alkyl: The term “lower alkyl” refers to a C₁-C₄ straight orbranched alkyl group. Exemplary lower alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term “lower haloalkyl” refers to a C₁-C₄ straightor branched alkyl group that is substituted with one or more halogenatoms.

Optionally substituted: As described herein, compounds of the inventionmay contain “optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen; —(CH₂)₀₋₄Rº;—(CH₂)₀₋₄ORº; —O(CH₂)₀₋₄Rº, —O—(CH₂)₀₋₄C(O)ORº; —(CH₂)₀₋₄CH(ORº)₂;—(CH₂)₀₋₄SRº; —(CH₂)₀₋₄Ph, which may be substituted with Rº;—(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substituted with Rº; —CH═CHPh, whichmay be substituted with Rº; —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may besubstituted with Rº; —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(Rº)₂;—(CH₂)₀₋₄N(Rº)C(O)Rº; —N(Rº)C(S)Rº; —(CH₂)₀₋₄N(Rº)C(O)NRº₂;)—N(Rº)C(S)NRº₂; —(CH₂)₀₋₄N(Rº)C(O)ORº; —N(Rº)N(Rº)C(O)Rº;—N(Rº)N(Rº)C(O)NRº₂; —N(Rº)N(Rº)C(O)ORº; —(CH₂)₀₋₄C(O)Rº; —C(S)Rº;—(CH₂)₀₋₄C(O)ORº; —(CH₂)₀₋₄C(O)SRº; —(CH₂)₀₋₄C(O)OSiRº₃;—(CH₂)₀₋₄OC(O)Rº; —OC(O)(CH₂)₀₋₄SR, —SC(S)SRº; —(CH₂)₀₋₄SC(O)Rº;—(CH₂)₀₋₄C(O)NRº₂; —C(S)NRº₂; —C(S)SRº; —SC(S)SRº, —(CH₂)₀₋₄OC(O)NRº₂;—C(O)N(ORº)Rº; —C(O)C(O)Rº; —C(O)CH₂C(O)Rº; —C(NORº)Rº; —(CH₂)₀₋₄SSRº;—(CH₂)₀₋₄S(O)₂Rº; —(CH₂)₀₋₄S(O)₂ORº; —(CH₂)₀₋₄OS(O)₂Rº; S(O)₂NRº₂;—(CH₂)₀₋₄S(O)Rº; —N(Rº)S(O)₂NRº₂; —N(Rº)S(O)₂Rº; —N(ORº)Rº; —C(NH)NRº₂;—P(O)₂Rº; —P(O)Rº₂; —OP(O)Rº₂; —OP(O)(ORº)₂; —SiRº₃; —(C₁₋₄ straight orbranched alkylene)O—N(Rº)₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(Rº)₂, wherein each Rº may be substituted as definedbelow and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences of Rº,taken together with their intervening atom(s), form a 3-12 memberedsaturated, partially unsaturated, or aryl mono- or bicyclic ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,which may be substituted as defined below.

Suitable monovalent substituents on Rº (or the ring formed by taking twoindependent occurrences of Rº together with their intervening atoms),are independently halogen, —(CH₂)₀₋₂R^(●), —(haloR^(●)), —(CH₂)₀₋₂OH,—(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN, —N₃,—(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(●) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), —(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12 membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), —(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Oral: The phrases “oral administration” and “administered orally” asused herein have their art-understood meaning referring toadministration by mouth of a compound or composition.

Parenteral: The phrases “parenteral administration” and “administeredparenterally” as used herein have their art-understood meaning referringto modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal, and intrasternal injection and infusion.

Partially unsaturated: As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptablesalt”, as used herein, refers to salts of such compounds that areappropriate for use in pharmaceutical contexts, i.e., salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge, et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 66: 1-19 (1977). In some embodiments, pharmaceuticallyacceptable salt include, but are not limited to, nontoxic acid additionsalts, which are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. In someembodiments, pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. In someembodiments, pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

Prodrug: A general, a “prodrug,” as that term is used herein and as isunderstood in the art, is an entity that, when administered to anorganism, is metabolized in the body to deliver an active (e.g.,therapeutic or diagnostic) agent of interest. Typically, such metabolisminvolves removal of at least one “prodrug moiety” so that the activeagent is formed. Various forms of “prodrugs” are known in the art. Forexamples of such prodrug moieties, see:

-   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and    Methods in Enzymology, 42:309-396, edited by K. Widder, et al.    (Academic Press, 1985);-   b) Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley,    2011);-   c) Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley,    2011);-   d) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen;-   e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H.    Bundgaard, p. 113-191 (1991);-   f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);-   g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285    (1988); and-   h) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

As with other compounds described herein, prodrugs may be provided inany of a variety of forms, e.g., crystal forms, salt forms etc. In someembodiments, prodrugs are provided as pharmaceutically acceptable saltsthereof.

Protecting group: The term “protecting group,” as used herein, is wellknown in the art and includes those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd)edition, John Wiley & Sons, 1999, the entirety of which is incorporatedherein by reference. Also included are those protecting groups speciallyadapted for nucleoside and nucleotide chemistry described in CurrentProtocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al.06/2012, the entirety of Chapter 2 is incorporated herein by reference.Suitable amino-protecting groups include methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD—Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′ - and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2(1,3-dithianyl)]methyl carbamate(Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc),2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-di methylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate,1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonami (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitably protected carboxylic acids further include, but are not limitedto, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylicacids. Examples of suitable silyl groups include trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triisopropylsilyl, and the like. Examples of suitable alkyl groupsinclude methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl,t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groupsinclude allyl. Examples of suitable aryl groups include optionallysubstituted phenyl, biphenyl, or naphthyl. Examples of suitablearylalkyl groups include optionally substituted benzyl (e.g.,p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.

Suitable hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tripxylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxyl-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3tetramethylbutyl)phenoxyacetate,2,4bis(1,1dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2methyl-2butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate,methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). Forprotecting 1,2- or 1,3-diols, the protecting groups include methyleneacetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylideneketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylideneacetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal,cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal,2,4dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal,2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethyleneacetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester,1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester,α-methoxybenzylidene ortho ester, 1-(N,N-dimethylamino)ethylidenederivative, α-(N,N′-dimethylamino)benzylidene derivative,2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

In some embodiments, a hydroxyl protecting group is acetyl, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl),4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl,trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate,tosylate, triflate, trityl, monomethoxytrityl (MMTr),4,4′-dimethoxytrityl, (DMTr) and 4,4′,4″-trimethoxytrityl (TMTr),2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE),2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl(NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl,2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl,2-(2-nitrophenyl)ethyl, butylthiocarbonyl,4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2- (dibromom ethyl)b enzoyl (Dbmb), 2-(i sopropylthiomethoxymethyl)benzoyl (Ptmt),9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).In some embodiments, each of the hydroxyl protecting groups is,independently selected from acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl and 4,4′-dimethoxytrityl. In some embodiments, thehydroxyl protecting group is selected from the group consisting oftrityl, monomethoxytrityl and 4,4′-dimethoxytrityl group.

In some embodiments, a phosphorous protecting group is a group attachedto the internucleotide phosphorous linkage throughout oligonucleotidesynthesis. In some embodiments, the phosphorous protecting group isattached to the sulfur atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorous protecting group isattached to the oxygen atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorous protecting group isattached to the oxygen atom of the internucleotide phosphate linkage. Insome embodiments the phosphorous protecting group is 2-cyanoethyl (CE orCne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl,benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe),2-phenylethyl, 3-(N-tert-butyl carboxamido)-1-propyl, 4-oxopentyl,4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl,3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl,2-(N-formyl, N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). In some embodiments, proteins include onlynaturally-occurring amino acids. In some embodiments, proteins includeone or more non-naturally-occurring amino acids (e.g., moieties thatform one or more peptide bonds with adjacent amino acids). In someembodiments, one or more residues in a protein chain contain anon-amino-acid moiety (e.g., a glycan, etc). In some embodiments, aprotein includes more than one polypeptide chain, for example linked byone or more disulfide bonds or associated by other means. In someembodiments, proteins contain L-amino acids, D-amino acids, or both; insome embodiments, proteins contain one or more amino acid modificationsor analogs known in the art. Useful modifications include, e.g.,terminal acetylation, amidation, methylation, etc. The term “peptide” isgenerally used to refer to a polypeptide having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids. In some embodiments, proteinsare antibodies, antibody fragments, biologically active portionsthereof, and/or characteristic portions thereof.

Sample: A “sample” as used herein is a specific organism or materialobtained therefrom. In some embodiments, a sample is a biological sampleobtained or derived from a source of interest, as described herein, . Insome embodiments, a source of interest comprises an organism, such as ananimal or human. In some embodiments, a biological sample comprisesbiological tissue or fluid. In some embodiments, a biological sample isor comprises bone marrow; blood; blood cells; ascites; tissue or fineneedle biopsy samples; cell-containing body fluids; free floatingnucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritonealfluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs;vaginal swabs; oral swabs; nasal swabs; washings or lavages such as aductal lavages or broncheoalveolar lavages; aspirates; scrapings; bonemarrow specimens; tissue biopsy specimens; surgical specimens; feces,other body fluids, secretions, and/or excretions; and/or cellstherefrom, etc. In some embodiments, a biological sample is or comprisescells obtained from an individual. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “processed sample” may comprise, for example nucleicacids or proteins extracted from a sample or obtained by subjecting aprimary sample to techniques such as amplification or reversetranscription of mRNA, isolation and/or purification of certaincomponents, etc. In some embodiments, a sample is an organism. In someembodiments, a sample is a plant. In some embodiments, a sample is ananimal. In some embodiments, a sample is a human. In some embodiments, asample is an organism other than a human.

Stereochemically isomeric forms: The phrase “stereochemically isomericforms,” as used herein, refers to different compounds made up of thesame atoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable. In someembodiments of the invention, provided chemical compositions may be orinclude pure preparations of individual stereochemically isomeric formsof a compound; in some embodiments, provided chemical compositions maybe or include mixtures of two or more stereochemically isomeric forms ofthe compound. In certain embodiments, such mixtures contain equalamounts of different stereochemically isomeric forms; in certainembodiments, such mixtures contain different amounts of at least twodifferent stereochemically isomeric forms. In some embodiments, achemical composition may contain all diastereomers and/or enantiomers ofthe compound. In some embodiments, a chemical composition may containless than all diastereomers and/or enantiomers of a compound. In someembodiments, if a particular enantiomer of a compound of the presentinvention is desired, it may be prepared, for example, by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, diastereomeric saltsare formed with an appropriate optically-active acid, and resolved, forexample, by fractional crystallization.

Subject: As used herein, the term “subject” or “test subject” refers toany organism to which a provided compound or composition is administeredin accordance with the present invention e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.) and plants. In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and/or chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with and/or displays oneor more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administeredsystemically,” “peripheral administration,” and “administeredperipherally” as used herein have their art-understood meaning referringto administration of a compound or composition such that it enters therecipient's system.

Tautomeric forms: The phrase “tautomeric forms,” as used herein, is usedto describe different isomeric forms of organic compounds that arecapable of facile interconversion. Tautomers may be characterized by theformal migration of a hydrogen atom or proton, accompanied by a switchof a single bond and adjacent double bond. In some embodiments,tautomers may result from prototropic tautomerism (i.e., the relocationof a proton). In some embodiments, tautomers may result from valencetautomerism (i.e., the rapid reorganization of bonding electrons). Allsuch tautomeric forms are intended to be included within the scope ofthe present invention. In some embodiments, tautomeric forms of acompound exist in mobile equilibrium with each other, so that attemptsto prepare the separate substances results in the formation of amixture. In some embodiments, tautomeric forms of a compound areseparable and isolatable compounds. In some embodiments of theinvention, chemical compositions may be provided that are or includepure preparations of a single tautomeric form of a compound. In someembodiments of the invention, chemical compositions may be provided asmixtures of two or more tautomeric forms of a compound. In certainembodiments, such mixtures contain equal amounts of different tautomericforms; in certain embodiments, such mixtures contain different amountsof at least two different tautomeric forms of a compound. In someembodiments of the invention, chemical compositions may contain alltautomeric forms of a compound. In some embodiments of the invention,chemical compositions may contain less than all tautomeric forms of acompound. In some embodiments of the invention, chemical compositionsmay contain one or more tautomeric forms of a compound in amounts thatvary over time as a result of interconversion. In some embodiments ofthe invention, the tautomerism is keto-enol tautomerism. One of skill inthe chemical arts would recognize that a keto-enol tautomer can be“trapped” (i.e., chemically modified such that it remains in the “enol”form) using any suitable reagent known in the chemical arts in toprovide an enol derivative that may subsequently be isolated using oneor more suitable techniques known in the art. Unless otherwiseindicated, the present invention encompasses all tautomeric forms ofrelevant compounds, whether in pure form or in admixture with oneanother.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the disease, disorder, and/orcondition, for example for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

Unsaturated: The term “unsaturated,” as used herein, means that a moietyhas one or more units of unsaturation.

Unit dose: The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Wild-type: As used herein, the term “wild-type” has its art-understoodmeaning that refers to an entity having a structure and/or activity asfound in nature in a “normal” (as contrasted with mutant, diseased,altered, etc) state or context. Those of ordinary skill in the art willappreciate that wild type genes and polypeptides often exist in multipledifferent forms (e.g., alleles).

Nucleic acid: The term “nucleic acid” includes any nucleotides, analogsthereof, and polymers thereof. The term “polynucleotide” as used hereinrefer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecules and, thus, include double- andsingle- stranded DNA, and double- and single-stranded RNA. These termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated, protected and/or capped nucleotides orpolynucleotides. The terms encompass poly- or oligo-ribonucleotides(RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derivedfrom N-glycosides or C-glycosides of nucleobases and/or modifiednucleobases; nucleic acids derived from sugars and/or modified sugars;and nucleic acids derived from phosphate bridges and/or modifiedphosphorus-atom bridges (also referred to herein as “internucleotidelinkages”). The term encompasses nucleic acids containing anycombinations of nucleobases, modified nucleobases, sugars, modifiedsugars, phosphate bridges or modified phosphorus atom bridges. Examplesinclude, and are not limited to, nucleic acids containing ribosemoieties, the nucleic acids containing deoxy-ribose moieties, nucleicacids containing both ribose and deoxyribose moieties, nucleic acidscontaining ribose and modified ribose moieties. The prefix poly- refersto a nucleic acid containing 2 to about 10,000 nucleotide monomer unitsand wherein the prefix oligo- refers to a nucleic acid containing 2 toabout 200 nucleotide monomer units.

Nucleotide: The term “nucleotide” as used herein refers to a monomericunit of a polynucleotide that consists of a heterocyclic base, a sugar,and one or more phosphate groups or phosphorus-containinginternucleotidic linkages. The naturally occurring bases, (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) arederivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleotides are linked via internucleotidiclinkages to form nucleic acids, or polynucleotides. Manyinternucleotidic linkages are known in the art (such as, though notlimited to, phosphate, phosphorothioates, boranophosphates and thelike). Artificial nucleic acids include PNAs (peptide nucleic acids),phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates,boranophosphates, methylphosphonates, phosphonoacetates,thiophosphonoacetates and other variants of the phosphate backbone ofnative nucleic acids, such as those described herein.

Nucleoside: The term “nucleoside” refers to a moiety wherein anucleobase or a modified nucleobase is covalently bound to a sugar ormodified sugar.

Sugar: The term “sugar” refers to a monosaccharide in closed and/or openform. Sugars include, but are not limited to, ribose, deoxyribose,pentofuranose, pentopyranose, and hexopyranose moieties. As used herein,the term also encompasses structural analogs used in lieu ofconventional sugar molecules, such as glycol, polymer of which forms thebackbone of the nucleic acid analog, glycol nucleic acid (“GNA”).

Modified sugar: The term “modified sugar” refers to a moiety that canreplace a sugar. The modified sugar mimics the spatial arrangement,electronic properties, or some other physicochemical property of asugar.

Nucleobase: The term “nucleobase” refers to the parts of nucleic acidsthat are involved in the hydrogen-bonding that binds one nucleic acidstrand to another complementary strand in a sequence specific manner.The most common naturally-occurring nucleobases are adenine (A), guanine(G), uracil (U), cytosine (C), and thymine (T). In some embodiments, thenaturally-occurring nucleobases are modified adenine, guanine, uracil,cytosine, or thymine. In some embodiments, the naturally-occurringnucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, a nucleobase is a “modified nucleobase,”e.g., a nucleobase other than adenine (A), guanine (G), uracil (U),cytosine (C), and thymine (T). In some embodiments, the modifiednucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, the modified nucleobase mimics the spatialarrangement, electronic properties, or some other physicochemicalproperty of the nucleobase and retains the property of hydrogen-bondingthat binds one nucleic acid strand to another in a sequence specificmanner. In some embodiments, a modified nucleobase can pair with all ofthe five naturally occurring bases (uracil, thymine, adenine, cytosine,or guanine) without substantially affecting the melting behavior,recognition by intracellular enzymes or activity of the oligonucleotideduplex.

Chiral ligand: The term “chiral ligand” or “chiral auxiliary” refers toa moiety that is chiral and can be incorporated into a reaction so thatthe reaction can be carried out with certain stereoselectivity.

Condensing reagent: In a condensation reaction, the term “condensingreagent” refers to a reagent that activates a less reactive site andrenders it more susceptible to attack by another reagent. In someembodiments, such another reagent is a nucleophile.

Blocking group: The term “blocking group” refers to a group that masksthe reactivity of a functional group. The functional group can besubsequently unmasked by removal of the blocking group. In someembodiments, a blocking group is a protecting group.

Moiety: The term “moiety” refers to a specific segment or functionalgroup of a molecule. Chemical moieties are often recognized chemicalentities embedded in or appended to a molecule.

Solid support: The term “solid support” refers to any support whichenables synthesis of nucleic acids. In some embodiments, the term refersto a glass or a polymer, that is insoluble in the media employed in thereaction steps performed to synthesize nucleic acids, and is derivatizedto comprise reactive groups. In some embodiments, the solid support isHighly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). Insome embodiments, the solid support is Controlled Pore Glass (CPG). Insome embodiments, the solid support is hybrid support of Controlled PoreGlass (CPG) and Highly Cross-linked Polystyrene (HCP).

Linking moiety: The term “linking moiety” refers to any moietyoptionally positioned between the terminal nucleoside and the solidsupport or between the terminal nucleoside and another nucleoside,nucleotide, or nucleic acid.

DNA molecule: A “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form or a double-stranded helix. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble- stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences can be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

Coding sequence: A DNA “coding sequence” or “coding region” is adouble-stranded DNA sequence which is transcribed and translated into apolypeptide in vivo when placed under the control of appropriateexpression control sequences. The boundaries of the coding sequence (the“open reading frame” or “ORF”) are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequencesfrom eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence is,usually, be located 3′ to the coding sequence. The term “non-codingsequence” or “non-coding region” refers to regions of a polynucleotidesequence that are not translated into amino acids (e.g. 5′ and 3′un-translated regions).

Reading frame: The term “reading frame” refers to one of the sixpossible reading frames, three in each direction, of the double strandedDNA molecule. The reading frame that is used determines which codons areused to encode amino acids within the coding sequence of a DNA molecule.

Antisense: As used herein, an “antisense” nucleic acid moleculecomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule, complementary to an mRNAsequence or complementary to the coding strand of a gene. Accordingly,an antisense nucleic acid molecule can associate via hydrogen bonds to asense nucleic acid molecule.

Wobble position: As used herein, a “wobble position” refers to the thirdposition of a codon. Mutations in a DNA molecule within the wobbleposition of a codon, in some embodiments, result in silent orconservative mutations at the amino acid level. For example, there arefour codons that encode Glycine, i.e., GGU, GGC, GGA and GGG, thusmutation of any wobble position nucleotide, to any other nucleotideselected from A, U , C and G, does not result in a change at the aminoacid level of the encoded protein and, therefore, is a silentsubstitution.

Silent substitution: a “silent substitution” or “silent mutation” is onein which a nucleotide within a codon is modified, but does not result ina change in the amino acid residue encoded by the codon. Examplesinclude mutations in the third position of a codon, as well in the firstposition of certain codons such as in the codon “CGG” which, whenmutated to AGG, still encodes Arg.

Gene: The terms “gene,” “recombinant gene” and “gene construct” as usedherein, refer to a DNA molecule, or portion of a DNA molecule, thatencodes a protein or a portion thereof. The DNA molecule can contain anopen reading frame encoding the protein (as exon sequences) and canfurther include intron sequences. The term “intron” as used herein,refers to a DNA sequence present in a given gene which is not translatedinto protein and is found in some, but not all cases, between exons. Itcan be desirable for the gene to be operably linked to, (or it cancomprise), one or more promoters, enhancers, repressors and/or otherregulatory sequences to modulate the activity or expression of the gene,as is well known in the art.

Complementary DNA: As used herein, a “complementary DNA” or “cDNA”includes recombinant polynucleotides synthesized by reversetranscription of mRNA and from which intervening sequences (introns)have been removed.

Homology: “Homology” or “identity” or “similarity” refers to sequencesimilarity between two nucleic acid molecules. Homology and identity caneach be determined by comparing a position in each sequence which can bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base, then the molecules areidentical at that position; when the equivalent site occupied by thesame or a similar nucleic acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage ofhomology/similarity or identity refers to a function of the number ofidentical or similar nucleic acids at positions shared by the comparedsequences. A sequence which is “unrelated” or “non-homologous” sharesless than 40% identity, less than 35% identity, less than 30% identity,or less than 25% identity with a sequence described herein. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

In some embodiments, the term “homology” describes a mathematicallybased comparison of sequence similarities which is used to identifygenes with similar functions or motifs. The nucleic acid sequencesdescribed herein can be used as a “query sequence” to perform a searchagainst public databases, for example, to identify other family members,related sequences or homologs. In some embodiments, such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments,BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous tonucleic acid molecules of the invention. In some embodiments, to obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used (See www.ncbi.nlm.nih.gov).

Identity: As used herein, “identity” means the percentage of identicalnucleotide residues at corresponding positions in two or more sequenceswhen the sequences are aligned to maximize sequence matching, i.e.,taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well-known Smith Watermanalgorithm can also be used to determine identity.

Heterologous: A “heterologous” region of a DNA sequence is anidentifiable segment of DNA within a larger DNA sequence that is notfound in association with the larger sequence in nature. Thus, when theheterologous region encodes a mammalian gene, the gene can usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous codingsequence is a sequence where the coding sequence itself is not found innature (e.g., a cDNA where the genomic coding sequence contains intronsor synthetic sequences having codons or motifs different than theunmodified gene). Allelic variations or naturally-occurring mutationalevents do not give rise to a heterologous region of DNA as definedherein.

Transition mutation: The term “transition mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by another pyrimidine, or a purine (adenosine(A) or guanosine (G) is replaced by another purine.

Transversion mutation: The term “transversion mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by a purine (adenosine (A) or guanosine (G),or a purine is replaced by a pyrimidine.

Oligonucleotide: the term “oligonucleotide” refers to a polymer oroligomer of nucleotide monomers, containing any combination ofnucleobases, modified nucleobases, sugars, modified sugars, phosphatebridges, or modified phosphorus atom bridges (also referred to herein as“internucleotidic linkage”, defined further herein).

Oligonucleotides can be single-stranded or double-stranded. As usedherein, the term “oligonucleotide strand” encompasses a single-strandedoligonucleotide. A single-stranded oligonucleotide can havedouble-stranded regions and a double-stranded oligonucleotide can havesingle-stranded regions. Exemplary oligonucleotides include, but are notlimited to structural genes, genes including control and terminationregions, self-replicating systems such as viral or plasmid DNA,single-stranded and double-stranded siRNAs and other RNA interferencereagents (RNAi agents or iRNA agents), shRNA, antisenseoligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, U1 adaptors, triplex-formingoligonucleotides, G-quadruplex oligonucleotides, RNA activators,immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

Double-stranded and single-stranded oligonucleotides that are effectivein inducing RNA interference are also referred to as siRNA, RNAi agent,or iRNA agent, herein. In some embodiments, these RNA interferenceinducing oligonucleotides associate with a cytoplasmic multi-proteincomplex known as RNAi-induced silencing complex (RISC). In manyembodiments, single-stranded and double-stranded RNAi agents aresufficiently long that they can be cleaved by an endogenous molecule,e.g., by Dicer, to produce smaller oligonucleotides that can enter theRISC machinery and participate in RISC mediated cleavage of a targetsequence, e.g. a target mRNA.

Oligonucleotides of the present invention can be of various lengths. Inparticular embodiments, oligonucleotides can range from about 2 to about200 nucleotides in length. In various related embodiments,oligonucleotides, single-stranded, double-stranded, and triple-stranded, can range in length from about 4 to about 10 nucleotides, fromabout 10 to about 50 nucleotides, from about 20 to about 50 nucleotides,from about 15 to about 30 nucleotides, from about 20 to about 30nucleotides in length. In some embodiments, the oligonucleotide is fromabout 9 to about 39 nucleotides in length. In some embodiments, theoligonucleotide is at least 4 nucleotides in length. In someembodiments, the oligonucleotide is at least 5 nucleotides in length. Insome embodiments, the oligonucleotide is at least 6 nucleotides inlength. In some embodiments, the oligonucleotide is at least 7nucleotides in length. In some embodiments, the oligonucleotide is atleast 8 nucleotides in length. In some embodiments, the oligonucleotideis at least 9 nucleotides in length. In some embodiments, theoligonucleotide is at least 10 nucleotides in length. In someembodiments, the oligonucleotide is at least 11 nucleotides in length.In some embodiments, the oligonucleotide is at least 12 nucleotides inlength. In some embodiments, the oligonucleotide is at least 15nucleotides in length. In some embodiments, the oligonucleotide is atleast 20 nucleotides in length. In some embodiments, the oligonucleotideis at least 25 nucleotides in length. In some embodiments, theoligonucleotide is at least 30 nucleotides in length. In someembodiments, the oligonucleotide is a duplex of complementary strands ofat least 18 nucleotides in length. In some embodiments, theoligonucleotide is a duplex of complementary strands of at least 21nucleotides in length.

Internucleotidic linkage: As used herein, the phrase “internucleotidiclinkage” refers generally to the phosphorus-containing linkage betweennucleotide units of an oligonucleotide, and is interchangeable with“inter-sugar linkage” and “phosphorus atom bridge,” as used above andherein. In some embodiments, an internucleotidic linkage is aphosphodiester linkage, as found in naturally occurring DNA and RNAmolecules. In some embodiments, an internucleotidic linkage is a“modified internucleotidic linkage” wherein each oxygen atom of thephosphodiester linkage is optionally and independently replaced by anorganic or inorganic moiety. In some embodiments, such an organic orinorganic moiety is selected from but not limited to ═S, ═Se, ═NR′,—SR′, —SeR′, —N(R′)₂, B(R′)₃, —S—, —Se—, and —N(R′)—, wherein each R′ isindependently as defined and described below. In some embodiments, aninternucleotidic linkage is a phosphotriester linkage, phosphorothioatediester linkage

or modified phosphorothioate triester linkage. It is understood by aperson of ordinary skill in the art that the internucleotidic linkagemay exist as an anion or cation at a given pH due to the existence ofacid or base moieties in the linkage.

Unless otherwise specified, when used with an oligonucleotide sequence,each of s, s1, s2, s3, s4, s5, s6 and s7 independently represents thefollowing modified internucleotidic linkage as illustrated in Table 1,below.

TABLE 1 Exemplary Modified Internucleotidic Linkage. Sym- bol ModifiedInternucleotidic Linkage s

s1

s2

s3

s4

s5

s6

s7

s8

s9

s10

s11

s12

s13

s14

s15

s16

s17

s18

For instance, (Rp, Sp)-ATsCs1GA has 1) a phosphorothioateinternucleotidic linkage

between T and C; and 2) a phosphorothioate triester internucleotidiclinkage having the structure of

between C and G. Unless otherwise specified, the Rp/Sp designationspreceding an oligonucleotide sequence describe the configurations ofchiral linkage phosphorus atoms in the internucleotidic linkagessequentially from 5′ to 3′ of the oligonucleotide sequence. Forinstance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the “s” linkagebetween T and C has Rp configuration and the phosphorus in “s1” linkagebetween C and G has Sp configuration. In some embodiments, “All-(Rp)” or“All-(Sp)” is used to indicate that all chiral linkage phosphorus atomsin oligonucleotide have the same Rp or Sp configuration, respectively.For instance, All-(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ IDNO: 1) indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Rp configuration;All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 2)indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Sp configuration.

Oligonucleotide type: As used herein, the phrase “oligonucleotide type”is used to define an oligonucleotide that has a particular basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “—XLR¹” groups in formula I).Oligonucleotides of a common designated “type” are structurallyidentical to one another.

One of skill in the art will appreciate that synthetic methods of thepresent invention provide for a degree of control during the synthesisof an oligonucleotide strand such that each nucleotide unit of theoligonucleotide strand can be designed and/or selected in advance tohave a particular stereochemistry at the linkage phosphorus and/or aparticular modification at the linkage phosphorus, and/or a particularbase, and/or a particular sugar. In some embodiments, an oligonucleotidestrand is designed and/or selected in advance to have a particularcombination of stereocenters at the linkage phosphorus. In someembodiments, an oligonucleotide strand is designed and/or determined tohave a particular combination of modifications at the linkagephosphorus. In some embodiments, an oligonucleotide strand is designedand/or selected to have a particular combination of bases. In someembodiments, an oligonucleotide strand is designed and/or selected tohave a particular combination of one or more of the above structuralcharacteristics. The present invention provides compositions comprisingor consisting of a plurality of oligonucleotide molecules (e.g.,chirally controlled oligonucleotide compositions). In some embodiments,all such molecules are of the same type (i.e., are structurallyidentical to one another). In many embodiments, however, providedcompositions comprise a plurality of oligonucleotides of differenttypes, typically in pre-determined relative amounts.

Chiral control: As used herein, “chiral control” refers to an ability tocontrol the stereochemical designation of every chiral linkagephosphorus within an oligonucleotide strand. The phrase “chirallycontrolled oligonucleotide” refers to an oligonucleotide which exists ina single diastereomeric form with respect to the chiral linkagephosphorus.

Chirally controlled oligonucleotide composition: As used herein, thephrase “chirally controlled oligonucleotide composition” refers to anoligonucleotide composition that contains predetermined levels ofindividual oligonucleotide types. For instance, in some embodiments achirally controlled oligonucleotide composition comprises oneoligonucleotide type. In some embodiments, a chirally controlledoligonucleotide composition comprises more than one oligonucleotidetype. In some embodiments, a chirally controlled oligonucleotidecomposition comprises a mixture of multiple oligonucleotide types.Exemplary chirally controlled oligonucleotide compositions are describedfurther herein.

Chirally pure: as used herein, the phrase “chirally pure” is used todescribe a chirally controlled oligonucleotide composition in which allof the oligonucleotides exist in a single diastereomeric form withrespect to the linkage phosphorus.

Chirally uniform: as used herein, the phrase “chirally uniform” is usedto describe an oligonucleotide molecule or type in which all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, an oligonucleotide whose nucleotide units all have Rpstereochemistry at the linkage phosphorus is chirally uniform. Likewise,an oligonucleotide whose nucleotide units all have Sp stereochemistry atthe linkage phosphorus is chirally uniform.

Predetermined: By predetermined is meant deliberately selected, forexample as opposed to randomly occurring or achieved. Those of ordinaryskill in the art, reading the present specification, will appreciatethat the present invention provides new and surprising technologies thatpermit selection of particular oligonucleotide types for preparationand/or inclusion in provided compositions, and further permitscontrolled preparation of precisely the selected particular types,optionally in selected particular relative amounts, so that providedcompositions are prepared. Such provided compositions are“predetermined” as described herein. Compositions that may containcertain individual oligonucleotide types because they happen to havebeen generated through a process that cannot be controlled tointentionally generate the particular oligonucleotide types is not a“predetermined” composition. In some embodiments, a predeterminedcomposition is one that can be intentionally reproduced (e.g., throughrepetition of a controlled process).

Linkage phosphorus: as defined herein, the phrase “linkage phosphorus”is used to indicate that the particular phosphorus atom being referredto is the phosphorus atom present in the internucleotidic linkage, whichphosphorus atom corresponds to the phosphorus atom of a phosphodiesterof an internucleotidic linkage as occurs in naturally occurring DNA andRNA. In some embodiments, a linkage phosphorus atom is in a modifiedinternucleotidic linkage, wherein each oxygen atom of a phosphodiesterlinkage is optionally and independently replaced by an organic orinorganic moiety. In some embodiments, a linkage phosphorus atom is P*of formula I. In some embodiments, a linkage phosphorus atom is chiral.In some embodiments, a chiral linkage phosphorus atom is P* of formulaI.

P-modification: as used herein, the term “P-modification” refers to anymodification at the linkage phosphorus other than a stereochemicalmodification. In some embodiments, a P-modification comprises addition,substitution, or removal of a pendant moiety covalently attached to alinkage phosphorus. In some embodiments, the “P-modification” is -X-L—R¹wherein each of X, L and R¹ is independently as defined and describedherein and below.

Blockmer: the term “blockmer,” as used herein, refers to anoligonucleotide strand whose pattern of structural featurescharacterizing each individual nucleotide unit is characterized by thepresence of at least two consecutive nucleotide units sharing a commonstructural feature at the internucleotidic phosphorus linkage. By commonstructural feature is meant common stereochemistry at the linkagephosphorus or a common modification at the linkage phosphorus. In someembodiments, the at least two consecutive nucleotide units sharing acommon structure feature at the internucleotidic phosphours linkage arereferred to as a “block”.

In some embodiments, a blockmer is a “stereoblockmer,” e.g., at leasttwo consecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. Such at lest two consecutive nucleotide units form a“stereoblock.” For instance, (Sp, Sp)-ATsCs1GA is a stereoblockmerbecause at least two consecutive nucleotide units, the Ts and the Cs1,have the same stereochemistry at the linkage phosphorus (both Sp). Inthe same oligonucleotide (Sp, Sp)-ATsCs1GA, TsCs1 forms a block, and itis a stereoblock.

In some embodiments, a blockmer is a “P-modification blockmer,” e.g., atleast two consecutive nucleotide units have the same modification at thelinkage phosphorus. Such at lest two consecutive nucleotide units form a“P-modification block”. For instance, (Rp, Sp)-ATsCsGA is aP-modification blockmer because at least two consecutive nucleotideunits, the Ts and the Cs, have the same P-modification (i.e., both are aphosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, TsCs forms a block, and it is a P-modification block.

In some embodiments, a blockmer is a “linkage blockmer,” e.g., at leasttwo consecutive nucleotide units have identical stereochemistry andidentical modifications at the linkage phosphorus. At least twoconsecutive nucleotide units form a “linkage block”. For instance, (Rp,Rp)-ATsCsGA is a linkage blockmer because at least two consecutivenucleotide units, the Ts and the Cs, have the same stereochemistry (bothRp) and P-modification (both phosphorothioate). In the sameoligonucleotide of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is alinkage block.

In some embodiments, a blockmer comprises one or more blocksindependently selected from a stereoblock, a P-modification block and alinkage block. In some embodiments, a blockmer is a stereoblockmer withrespect to one block, and/or a P-modification blockmer with respect toanother block, and/or a linkage blockmer with respect to yet anotherblock. For instance, (Rp, Rp, Rp, Rp, Rp, Sp, Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG (SEQ ID NO: 3) is a stereoblockmer withrespect to the stereoblock AsTsCsGsAs1 (all Rp at linkage phosphorus) orTs1Cs1Gs1 (all Sp at linkage phosphorus), a P-modification blockmer withrespect to the P-modification block AsTsCsGs (all s linkage) orAs1Ts1Cs1Gs1 (all s1 linkage), or a linkage blockmer with respect to thelinkage block AsTsCsGs (all Rp at linkage phosphorus and all s linkage)or Ts1Cs1Gs1 (all Sp at linkage phosphorus and all s1 linkage).

Altmer: the term “altmer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is characterized in that no two consecutivenucleotide units of the oligonucleotide strand share a particularstructural feature at the internucleotidic phosphorus linkage. In someembodiments, an altmer is designed such that it comprises a repeatingpattern. In some embodiments, an altmer is designed such that it doesnot comprise a repeating pattern.

In some embodiments, an altmer is a “stereoaltmer,” e.g., no twoconsecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 4).

In some embodiments, an altmer is a “P-modification altmer” e.g., no twoconsecutive nucleotide units have the same modification at the linkagephosphorus. For instance, All-(Sp)-CAs1GsT, in which each linkagephosphorus has a different P-modification than the others.

In some embodiments, an altmer is a “linkage altmer,” e.g., no twoconsecutive nucleotide units have identical stereochemistry or identicalmodifications at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC (SEQ ID NO: 5).

Unimer: the term “unimer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is such that all nucleotide units within thestrand share at least one common structural feature at theinternucleotidic phosphorus linkage. By common structural feature ismeant common stereochemistry at the linkage phosphorus or a commonmodification at the linkage phosphorus.

In some embodiments, a unimer is a “stereounimer,” e.g., all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, All-(Sp)-CsAs1GsT, in which all the linkages have Spphosphorus.

In some embodiments, a unimer is a “P-modification unimer”, e.g., allnucleotide units have the same modification at the linkage phosphorus.For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO:6), in which all the internucleotidic linkages are phosphorothioatediester.

In some embodiments, a unimer is a “linkage unimer,” e.g., allnucleotide units have the same stereochemistry and the samemodifications at the linkage phosphorus. For instance,All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 7), inwhich all the internucleotidic linkages are phosphorothioate diesterhaving Sp linkage phosphorus.

Gapmer: as used herein, the term “gapmer” refers to an oligonucleotidestrand characterized in that at least one internucleotidic phosphoruslinkage of the oligonucleotide strand is a phosphate diester linkage,for example such as those found in naturally occurring DNA or RNA. Insome embodiments, more than one internucleotidic phosphorus linkage ofthe oligonucleotide strand is a phosphate diester linkage such as thosefound in naturally occurring DNA or RNA. For instance, All-(Sp)-CAs1GsT,in which the internucleotidic linkage between C and A is a phosphatediester linkage.

Skipmer: as used herein, the term “skipmer” refers to a type of gapmerin which every other internucleotidic phosphorus linkage of theoligonucleotide strand is a phosphate diester linkage, for example suchas those found in naturally occurring DNA or RNA, and every otherinternucleotidic phosphorus linkage of the oligonucleotide strand is amodified internucleotidic linkage. For instance,All-(Sp)-AsTCs1GAs2TCs3G.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The methods and structures described herein relating to compounds andcompositions of the invention also apply to the pharmaceuticallyacceptable acid or base addition salts and all stereoisomeric forms ofthese compounds and compositions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Reverse phase HPLCs after incubation with rat liver homogenate.Total amounts of oligonucleotides remaining when incubated with ratwhole liver homogenate at 37° C. at different days were measured. Thein-vitro metabolic stability of ONT-154 was found to be similar toONT-87 which has 2′-MOE wings while both have much better stability than2′-MOE gapmer which is stereorandom (ONT-41, Mipomersen). The amount offull length oligomer remaining was measured by reverse phase HPLC wherepeak area of the peak of interest was normalized with internal standard.

FIG. 2. Degradation of various chirally pure analogues of Mipomersen(ONT-41) in rat whole liver homogenate. Total amounts of oligonucleotideremaining when incubated with rat whole liver homogenate at 37° C. atdifferent days were measured. The in-vitro metabolic stability ofchirally pure diastereomers of human ApoB sequence ONT-41 (Mipomersen)was found to increase with increased Sp internucleotidic linkages. Theamount of full length oligomer remaining was measured by reverse phaseHPLC where peak area of the peak of interest was normalized withinternal standard.

FIG. 3. Degradation of various chirally pure analogues of mouse ApoBsequence (ISIS 147764, ONT-83) in rat whole liver homogenate. Totalamounts of oligonucleotide remaining when incubated with rat whole liverhomogenate at 37° C. at different days were measured. The in-vitrometabolic stability of chirally pure diastereomers of murine ApoBsequence (ONT-83, 2′-MOE gapmer, stereorandom phosphorothioate) wasfound to increase with increased Sp internucleotidic linkages. Theamount of full length oligomer remaining was measured by reverse phaseHPLC where peak area of the peak of interest was normalized withinternal standard.

FIG. 4. Degradation of Mipomersen analogue ONT-75 in rat whole liverhomogenate over a period of 24hrs. This figure illustrates stability ofONT-75 in rate whole liver homogenate.

FIG. 5. Degradation of Mipomersen analogue ONT-81 in rat whole liverhomogenate over a period of 24hrs. This figure illustrates stability ofONT-81 in rate whole liver homogenate.

FIG. 6. Durations of knockdown for ONT-87, ONT-88, and ONT-89.Stereoisomers can exhibit substantially different durations ofknockdown. ONT-87 results in substantially more durable suppression thanother stereoisomers. Increased duration of action of ONT-87 in multiplein vivo studies were observed. ONT-88 showed similar efficacy andrecovery profile as ONT-41 (Mipomersen) in certain in-vivo studies. HuApoB transgenic mice, n=4, were dosed with 10 mpk IP bolus, 2X/week forthree weeks. The mice were randomized to study groups, and dosedintraperitoneally (IP) at 10 mg/kg on Days 1, 4, 8, 11, 15, 18, and 22,based on individual mouse body weight measured prior to dosing on eachdosing day. Blood was collected on days 0, 17, 24, 31, 38, 45 and 52 bysubmandibular (cheek) bleed and at sacrifice on Day 52 by cardiacpuncture and then processed to serum. ApoB was measured by ELISA.Highlighted: 72% vs. 35% knock-down maintained at 3 weeks postdose.

FIG. 7. HPLC profiles exhibiting the difference in metabolic stabilitydetermined in Human Serum for siRNA duplexes having several Rp, Sp orstereorandom phosphorothioate linkages.

FIG. 8. Effect of stereochemistry on RNase H activity. Oligonucleotideswere hybridized with RNA and then incubated with RNase H at 37° C. inthe presence of 1X RNase H buffer. From top to bottom at 120 min:ONT-89, ONT-77, ONT-81, ONT-80, ONT-75, ONT-41, ONT-88, ONT-154, ONT-87,with ONT-77/154 very close to each other.

FIG. 9. Analysis of human RNase H1 cleavage of a 20-mer RNA whenhybridized with different preparations of stereoisomers ofphosphorothioate oligonucleotides targeting the same region of humanApoB mRNA. Specific sites of cleavage are strongly influenced by thedistinct stereochemistries. Arrows represent position of cleavage(cleavage sites). Products were analyzed by UPLC/MS. The length of thearrow signifies the amount of products present in the reaction mixturewhich was determined from the ratio of UV peak area to theoreticalextinction coefficient of that fragment (the larger the arrow, the morethe detected cleavage products). (A): Legend for cleavage maps. (B) and(C): cleavage maps of oligonucleotides. ONT-41 (SEQ ID NO: 239), ONT-75(SEQ ID NO: 244), ONT-77 (SEQ ID NO: 245), ONT-80 (SEQ ID NO: 246),ONT-81 (SEQ ID NO: 247), ONT-87 (SEQ ID NO: 248), ONT-88 (SEQ ID NO:249), ONT-89 (SEQ ID NO: 250), and ONT-154 (SEQ ID NO: 241).

FIG. 10. Cleavage maps of different oligonucleotide compositions((A)-(C)). These three sequences target different regions in FOXO1 mRNA.Each sequence was studied with five different chemistries. Cleavage mapsare derived from reaction mixtures obtained after 30 minutes ofincubation of respective duplexes with RNase H1C in the presence of1XPBS buffer at 37° C. Arrows indicate sites of cleavage. (

) indicates that both fragments, 5′-phosphate specie as well as 5′-OH3′-OH specie were identified in reaction mixtures. (

) indicates that only 5′-phosphate specie was detected and (

) indicates that 5′-OH 3′-OH component was detected in mass spectrometryanalysis. The length of the arrow signifies the amount of productspresent in the reaction mixture which was determined from the ratio ofUV peak area to theoretical extinction coefficient of that fragment (thelarger the arrow, the more the detectable cleavage products). Only inthe cases where 5′-OH 3′-OH was not detected in the reaction mixture,5′-phosphate specie peak was used for quantification. Cleavage rateswere determined by measuring amount of full length RNA remaining in thereaction mixtures by reverse phase HPLC. Reactions were quenched atfixed time points by 30 mM Na₂EDTA. ONT-316 (SEQ ID NO: 516), ONT-355(SEQ ID NO: 517), ONT-361 (SEQ ID NO: 518), ONT-367 (SEQ ID NO: 519),ONT-373 (SEQ ID NO: 520), ONT-302 (SEQ ID NO: 522), ONT-352 (SEQ ID NO:523), ONT-358 (SEQ ID NO: 524), ONT-364 (SEQ ID NO: 525), ONT-370 (SEQID NO: 526), ONT-315 (SEQ ID NO: 528), ONT-354 (SEQ ID NO: 529), ONT-360(SEQ ID NO: 530), ONT-366 (SEQ ID NO: 531), and ONT-372 (SEQ ID NO:532).

FIG. 11. Cleavage maps of oligonucleotide compositions having differentcommon base sequences and lengths ((A)-(B)). The maps show a comparisonof stereorandom DNA compositions (top panel) with three distinct andstereochemically pure oligonucleotide compositions. Data compare resultsof chirally controlled oligonucleotide compositions with twostereorandom phosphorothioate oligonucleotide compositions (ONT-366 andONT-367) targeting different regions in FOXO1 mRNA. Each panel shows acomparison of setreorandom DNA (top panel) with three distinct andstereochemically pure oligonucleotide preparaitons. Cleavage maps werederived from reaction mixtures obtained after 30 minutes of incubationof respective duplexes with RNase H1C in the presence of 1XPBS buffer at37° C. Arrows indicate sites of cleavage. (

) indicates that both fragments, 5′-phosphate specie as well as 5′-OH3′-OH specie were identified in reaction mixtures. (

) indicates that only 5′-phosphate specie was detected and (

) indicates that 5′-OH 3′-OH component was detected in mass spectrometryanalysis. The length of the arrow signifies the amount of metabolitepresent in the reaction mixture which was determined from the ratio ofUV peak area to theoretical extinction coefficient of that fragment (thelarger the arrow, the more the detectable cleavage products). Only inthe cases where 5′-OH 3′-OH was not detected in the reaction mixture,5′-phosphate specie peak was used for quantification. ONT-366 (SEQ IDNO: 531), ONT-389 (SEQ ID NO: 565), ONT-390 (SEQ ID NO: 566), ONT-391(SEQ ID NO: 567), ONT-367 (SEQ ID NO: 519), ONT-392 (SEQ ID NO: 568),ONT-393 (SEQ ID NO: 569), and ONT-394 (SEQ ID NO: 570).

FIG. 12. Effect of stereochemistry on RNase H activity. In twoindependent experiments, antisense oligonucleotides targeting anidentical region of FOXO1 mRNA were hybridized with RNA and thenincubated with RNase H at 37° C. in the presence of 1X RNase H buffer.Disappearance of full length RNA was measured from its peak area at 254nm using RP-HPLC. (A): from top to bottom at 60 min: ONT-355, ONT-316,ONT-367, ONT-392, ONT-393 and ONT-394 (ONT-393 and ONT-394 about thesame at 60 min; ONT-393 had higher %RNA substrate remaining at 5 min).(B): from top to bottom at 60 min: ONT-315, ONT-354, ONT-366, ONT-391,ONT-389 and ONT-390. Cleavage rates were determined by measuring amountof full length RNA remaining in the reaction mixtures by reverse phaseHPLC. Reactions were quenched at fixed time points by 30 mM Na₂EDTA.

FIG. 13. Turnover of antisense oligonucleotides. The duplexes were madewith each DNA strand concentration equal to 6μM and RNA being 100 μM.These duplexes were incubated with 0.02 μM RNase H enzyme anddisappearance of full length RNA was measured from its peak area at 254nm using RP-HPLC. Cleavage rates were determined by measuring amount offull length RNA remaining in the reaction mixtures by reverse phaseHPLC. Reactions were quenched at fixed time points by 30 mM Na₂EDTA.From top to bottom at 40 min: ONT-316, ONT-367 and ONT-392.

FIG. 14. Cleavage map comparing a stereorandom phosphorothioateoligonucleotide with six distinct and stereochemically pureoligonucleotide preparations targeting the same FOXO1 mRNA region.ONT-367 (SEQ ID NO: 519), ONT-392 (SEQ ID NO: 568), ONT-393 (SEQ ID NO:569), ONT-394 (SEQ ID NO: 570), ONT-400 (SEQ ID NO: 575), ONT-401 (SEQID NO: 576), and ONT-406 (SEQ ID NO: 581).

FIG. 15. Effect of stereochemistry on RNase H activity. Antisenseoligonucleotides were hybridized with RNA and then incubated with RNaseH at 37° C. in the presence of 1×RNase H buffer. Dependence ofstereochemistry upon RNase H activity was observed. Also evident incomparing ONT-367 (stereorandom DNA) and ONT-316 (5-10-5 2′-MOE Gapmer)is the strong dependence of compositional chemistry upon RNase Hactivity. From top to bottom at 40 min: ONT-316, ONT-421, ONT-367,ONT-392, ONT-394, ONT-415, and ONT-422 (ONT-394/415/422 have similarlevels at 40 min; at 5 min, ONT-422>ONT-394 >ONT-415 in % RNA remainingin DNA/RNA duplex).

FIG. 16. Effect of stereochemistry on RNase H activity. Antisenseoligonucleotides targeting an identical region of FOXO1 mRNA werehybridized with RNA and then incubated with RNase H at 37° C. in thepresence of 1X RNase H buffer. Dependence of stereochemistry upon RNaseH activity was observed. Form top to bottom at 40 min: ONT-396, ONT-409,ONT-414, ONT-408 (ONT-396/409/414/408 have similar levels at 40 min),ONT-404, ONT-410, ONT-402 (ONT-404/410/408 have similar levels at 40min), ONT-403, ONT-407, ONT-405, ONT-401, ONT-406 and ONT-400(ONT-401/405/406/400 have similar levels at 40 min).

FIG. 17. Effect of stereochemistry on RNase H activity. Antisenseoligonucleotides targeting an identical region of FOXO1 mRNA werehybridized with RNA and then incubated with RNase H at 37° C. in thepresence of 1X RNase H buffer. Dependence of stereochemistry upon RNaseH activity was observed. ONT-406 was observed to elicit cleavage ofduplexed RNA at a rate in slight excess of that of the phosphodiesteroligonucleotide ONT-415. From top to bottom at 40 min: ONT-396, ONT-421,ONT-392, ONT-394, ONT-415 ONT-406, and ONT-422 (ONT-394/415/406 havesimilar levels at 40 min; at 5 min, ONT-394>ONT-415>ONT-406 in % RNAremaining in DNA/RNA duplex).

FIG. 18. Exemplary UV chromatograms of RNA cleavage products obtainedwhen RNA (ONT-388) was duplexed with stereorandom DNA, ONT-367 (top) andstereopure DNA with repeat triplet motif-3′-SSR-5′, ONT-394 (bottom).).2.35 min: 7 mer; 3.16 min: 8 mer and p-6 mer; 4.48 min: P-7 mer; 5.83min: P-8 mer; 6.88 min: 12 mer; 9.32 min: 13 mer; 10.13 min: P-11 mer;11.0 min: P-12 mer and 14 mer; 11.93 min: P-13 mer; 13.13 min: P-14 mer.ONT-394 (on the bottom) peak assignment: 4.55min: p-7mer; 4.97min:lOmer; 9.53min: 13mer.

FIG. 19. Electrospray Ionization Spectrum of RNA cleavage products. RNAfragments obtained from the duplex ONT-387, RNA/ONT-354, (7-6-7,DNA-2′-OMe-DNA) on the top and ONT-387, RNA/ONT-315, (5-10-5,2′-MOEGapmer) at the bottom when these duplexes were incubated with RNase Hfor 30 min in the presence of 1X RNse H buffer.

FIG. 20. UV Chromatogram and TIC of ONT-406 and ONT-388 duplex after 30minutes of incubation with RNase H.

FIG. 21. An exemplary proposed cleavage. Provided chirally controlledoligonucleotide compositions are capable of cleaving targets asdepicted.

FIG. 22. Exemplary allele specific cleavage targeting mutant HuntingtinmRNA. (A) and (B): exemplary oligonucleotides. (C)-(E): cleavage maps.(F)-(H): RNA cleavage. Stereorandom and chirally controlledoligonucleotide compositions were prepared to target single nucleotidepolymorphisms for allele selective suppression of mutant Huntingtin.ONT-450 (stereorandom) targeting ONT-453 (muHTT) and ONT-454 (wtHTT)showed marginal differentiation in RNA cleavage and their cleavage maps.Chirally controlled ONT-451 with selective placement of 3′-SSR-5′ motifin RNase H recognition site targeting ONT-453 (muHTT) and ONT-454(wtHTT) showed large differentiation in RNA cleavage rate. From thecleavage map, it is notable that 3′-SSR-5′ motif is placed to direct thecleavage between positions 8 and 9 which is after the mismatch if readfrom 5′-end of RNA. ONT-452 with selective placement of 3′-SSR-5′ motifin RNase H recognition site targeting ONT-453 (muHTT) and ONT-454(wtHTT) showed moderate differentiation in RNA cleavage rate. 3′-SSR-5′motif was placed to direct the cleavage at positions 7 and 8 which isbefore the mismatch if read from 5′-end of RNA. Exemplary dataillustrate significance of position in placement of 3′-SSR-5′ motif toachieve enhanced discrimination for allele specific cleavage. Allcleavage maps are derived from the reaction mixtures obtained after 5minutes of incubation of respective duplexes with RNase H1C in thepresence of 1XPBS buffer at 37° C. Arrows indicate sites of cleavage. (

) indicates that both fragments, 5′-phosphate specie as well as 5′—OH3′—OH specie were identified in reaction mixtures. (

) indicates that only 5′-phosphate specie was detected and (

) indicates that 5′—OH 3′—OH component was detected in mass spectrometryanalysis. The length of the arrow signifies the amount of metabolitepresent in the reaction mixture which was determined from the ratio ofUV peak area to theoretical extinction coefficient of that fragment.Only in the cases where 5′—OH 3′—OH was not detected in the reactionmixture, 5′-phosphate specie peak was used for quantification. ONT-450(SEQ ID NO: 555), ONT-451 (SEQ ID NO: 593), ONT-452 (SEQ ID NO: 594),ONT-453 (SEQ ID NO: 563), ONT-454 (SEQ ID NO: 564).

FIG. 23. (A)-(C): exemplary allele specific cleavage targeting FOXO1mRNA. ONT-388 (SEQ ID NO: 559), ONT-442 (SEQ ID NO: 561), ONT-443 (SEQID NO: 562), ONT-400 (SEQ ID NO: 575), ONT-402 (SEQ ID NO: 577), ONT-406(SEQ ID NO: 581).

FIG. 24. In vitro dose response silencing of ApoB mRNA after treatmentwith ApoB oligonucleotides. Stereochemically pure diasetereomers withand without 2′-MOE wings show similar efficacy as ONT-41 (Mipomersen).

FIG. 25. Comparison of RNase H cleavage maps (A) and RNA cleavage rates(B) for stereorandom composition (ONT-367) and chirally controlledoligonucleotide compositions (ONT-421, all Sp and ONT-455, all Rp) andDNA (ONT-415). These sequences target the same region in FOXO1 mRNA.Cleavage maps were derived from the reaction mixtures obtained after 5minutes of incubation of respective duplexes with RNase H1C in thepresence of 1XPBS buffer at 37° C. Arrows indicate sites of cleavage. (

) indicates that both fragments, 5′-phosphate specie as well as 5′-OH3′-OH specie were identified in reaction mixtures. (

) indicates that only 5′-phosphate specie was detected and (

) indicates that 5′-OH 3′-OH component was detected in mass spectrometryanalysis. The length of the arrow signifies the amount of metabolitepresent in the reaction mixture which was determined from the ratio ofUV peak area to theoretical extinction coefficient of that fragment.Only in the cases where 5′-OH 3′-OH was not detected in the reactionmixture, 5′-phosphate specie peak was used for quantification. Cleavagerates were determined by measuring amount of full length RNA remainingin the reaction mixtures by reverse phase HPLC. Reactions are quenchedat fixed time points by 30 mM Na₂EDTA. ONT-367 (SEQ ID NO: 519), ONT-421(SEQ ID NO: 590), ONT-455 (SEQ ID NO: 592), and ONT-415 (SEQ ID NO:560).

FIG. 26. Comparison of cleavage maps of sequences containing one Rp withchange of position starting from 3′-end of DNA. These sequences targetthe same region in FOXO1 mRNA. Cleavage maps are derived from thereaction mixtures obtained after 5 minutes of incubation of respectiveduplexes with RNase H1C in the presence of 1XPBS buffer at 37° C. Arrowsindicate sites of cleavage. (

) indicates that both fragments, 5′-phosphate specie as well as 5′—OH3′—OH specie were identified in reaction mixtures. (

) indicates that only 5′-phosphate specie was detected and (

) indicates that 5′—OH 3′—OH component was detected in mass spectrometryanalysis. The length of the arrow signifies the amount of metabolitepresent in the reaction mixture which was determined from the ratio ofUV peak area to theoretical extinction coefficient of that fragment.Only in the cases where 5′—OH 3′—OH was not detected in the reactionmixture, 5′-phosphate specie peak was used for quantification. ONT-396(SEQ ID NO: 571), ONT-397 (SEQ ID NO: 572), ONT-398 (SEQ ID NO: 573),ONT-399 (SEQ ID NO: 574), ONT-400 (SEQ ID NO: 575), ONT-401 (SEQ ID NO:576), ONT-402 (SEQ ID NO: 577), ONT-403 (SEQ ID NO: 578), ONT-404 (SEQID NO: 579), ONT-405 (SEQ ID NO: 580), ONT-406 (SEQ ID NO: 581), ONT-407(SEQ ID NO: 582), ONT-408 (SEQ ID NO: 583), ONT-409 (SEQ ID NO: 584),ONT-410 (SEQ ID NO: 585), ONT-411 (SEQ ID NO: 586), ONT-412 (SEQ ID NO:587), ONT-413 (SEQ ID NO: 588), and ONT-414 (SEQ ID NO: 589).

FIG. 27. (A) Comparison of RNase H cleavage rates for stereopureoligonucleotides (ONT-406), (ONT-401), (ONT-404) and (ONT-408). All foursequences are stereopure phosphorothioates with one Rp linkage. Thesesequences target the same region in FOXO1 mRNA. All duplexes wereincubation with RNase H1C in the presence of 1XPBS buffer at 37° C.Reactions were quenched at fixed time points by 30 mM Na₂EDTA. Cleavagerates were determined by measuring amount of full length RNA remainingin the reaction mixtures by reverse phase HPLC. ONT-406 and ONT-401 werefound to have superior cleavage rates. (B) Correlation between %RNAcleaved in RNase H assay (10 μM oligonucleotide) and %mRNA knockdown inin vitro assay (20 nM oligonucleotide). All sequences target the sameregion of mRNA in the FOXO1 target. The quantity of RNA remaining isdetermined by UV peak area for RNA when normalized to DNA in the samereaction mixture. All of the above maps are derived from the reactionmixture obtained after 5 minutes of incubation of respective duplexeswith RNase H1C in the presence of 1XPBS buffer at 37° C. All sequencesfrom ONT-396 to ONT-414 have one Rp phosphorothioate and they vary inthe position of Rp. ONT-421 (All Sp) phosphorothioate was inactivein-vitro assay. It relates poor cleavage rate of RNA in RNase H assaywhen ONT-421 is duplexed with complementary RNA.

FIG. 28. Serum stability assay of single Rp walk PS DNA(ONT-396-ONT-414), stereorandom PS DNA(ONT-367), all-Sp PS DNA (ONT-421)and all-Rp PS DNA (ONT-455) in rat serum for 2 days. Note ONT-396 andONT-455 decomposed at tested time point.

FIG. 29. Exemplary oligonucleotides including hemimers. (A): cleavagemaps. (B): RNA cleavage assay. (C): FOXO1 mRNA knockdown. In someembodiments, introduction of 2′-modifications on 5′-end of the sequencesincreases stability for binding to target RNA while maintaining RNase Hactivity. ONT-367 (stereorandom phosphorothioate DNA) and ONT-440 (5-15,2′-F-DNA) have similar cleavage maps and similar rate of RNA cleavage inRNase H assay (10 μM oligonucleotide). In some embodiments, ONT-440(5-11, 2′-F-DNA) sequence can have better cell penetration properties.In some embodiments, asymmetric 2′-modifications provide Tm advantagewhile maintaining RNase H activity. Introduction of RSS motifs canfurther enhance RNase H efficiency in the hemimers. Cleavage maps arederived from the reaction mixtures obtained after 5 minutes ofincubation of respective duplexes with RNase H1C in the presence of1XPBS buffer at 37° C. Arrows indicate sites of cleavage. (

) indicates that both fragments, 5′-phosphate specie as well as 5′-OH3′-OH specie were identified in reaction mixtures. (

) indicates that only 5′-phosphate specie was detected and (

) indicates that 5′—OH 3′—OH component was detected in mass spectrometryanalysis. The length of the arrow signifies the amount of metabolitepresent in the reaction mixture which was determined from the ratio ofUV peak area to theoretical extinction coefficient of that fragment.Only in the cases where 5′—OH 3′—OH was not detected in the reactionmixture, 5′-phosphate specie peak was used for quantification. ONT-367(SEQ ID NO: 511), ONT-440 (SEQ ID NO: 611), ONT-441 (SEQ ID NO: 612).

FIG. 30. Exemplary mass spectrometry data of cleavage assay. Top: datafor ONT-367: 2.35 min: 7 mer; 3.16 min: 8 mer and P-6 mer; 4.58 min: P-7mer; 5.91 min: P-8 mer; 7.19 min: 12 mer; 9.55 min: 13 mer; 10.13 min:P-11 mer; 11.14 min: P-12 mer and 14 mer; 12.11 min: P-13 mer; 13.29min: P-14 mer; 14.80 min: full length RNA (ONT-388) and 18.33 min:stereorandom DNA (ONT-367). Bottom: data for ONT-406: 4.72 min:p-rArUrGrGrCrUrA, 5′ -phosphorylated 7 mer RNA; 9.46 min: 5′-rGrUrGrArGrCrArGrCrUrGrCrA (SEQ ID NO: 8), 5′-OH 3′-OH 13 mer RNA;16.45 min: full length RNA (ONT-388); 19.48 and 19.49 min: stereopureDNA (ONT-406).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Synthetic oligonucleotides provide useful molecular tools in a widevariety of applications. For example, oligonucleotides are useful intherapeutic, diagnostic, research, and new nanomaterials applications.The use of naturally occurring nucleic acids (e.g., unmodified DNA orRNA) is limited, for example, by their susceptibility to endo- andexo-nucleases. As such, various synthetic counterparts have beendeveloped to circumvent these shortcomings. These include syntheticoligonucleotides that contain backbone modifications, which render thesemolecules less susceptible to degradation. From a structural point ofview, such modifications to internucleotide phosphate linkages introducechirality. It has become clear that certain properties ofoligonucleotides may be affected by the configurations of the phosphorusatoms that form the backbone of the oligonucleotides. For example, invitro studies have shown that the properties of antisense nucleotidessuch as binding affinity, sequence specific binding to the complementaryRNA, stability to nucleases are affected by, inter alia, chirality ofthe backbone (e.g., the configurations of the phosphorus atoms).

Among other things, the present invention encompasses the recognitionthat stereorandom oligonucleotide preparations contain a plurality ofdistinct chemical entities that differ from one another in thestereochemical structure of individual backbone chiral centers withinthe oligonucleotide chain. Moreover, the present invention encompassesthe insight that it is typically unlikely that a stereorandomoligonucleotide preparation will include every possible stereoisomer ofthe relevant oligonucleotide. Thus, among other things, the presentinvention provides new chemical entities that are particularstereoisomers of oligonucleotides of interest. That is, the presentinvention provides substantially pure preparations of singleoligonucleotide compounds, where a particular oligonucleotide compoundmay be defined by its base sequence, its length, its pattern of backbonelinkages, and its pattern of backbone chiral centers.

The present invention demonstrates, among other things, that individualstereoisomers of a particular oligonucleotide can show differentstability and/or activity from each other. Moreover, the presentdisclosure demonstrates that stability improvements achieved throughinclusion and/or location of particular chiral structures within anoligonucleotide can be comparable to, or even better than those achievedthrough use of modified backbone linkages, bases, and/or sugars (e.g.,through use of certain types of modified phosphates, 2′-modifications,base modifications, etc.). The present disclosure, in some embodiments,also demonstrates that activity improvements achieved through inclusionand/or location of particular chiral structures within anoligonucleotide can be comparable to, or even better than those achievedthrough use of modified backbone linkages, bases, and/or sugars (e.g.,through use of certain types of modified phosphates, 2′-modifications,base modifications, etc.). In some embodiments, inclusion and/orlocation of particular chiral linkages within an oligonucleotide cansurprisingly change the cleavage pattern of a nucleic acid polymer whensuch an oligonucleotide is utilized for cleaving said nucleic acidpolymer. For example, in some embodiments, a pattern of backbone chiralcenters provides unexpectedly high cleavage efficiency of a targetnucleic acid polymer. In some embodiments, a pattern of backbone chiralcenters provides new cleavage sites. In some embodiments, a pattern ofbackbone chiral centers provides fewer cleavage sites, for example, byblocking certain existing cleavage sites. Even more unexpectedly, insome embodiments, a pattern of backbone chiral centers provides cleavageat only one site of a target nucleic acid polymer within the sequencethat is complementary to a oligonucleotide utilized for cleavage. Insome embodiments, higher cleavage efficiency is achieved by selecting apattern of backbone chiral centers to minimize the number of cleavagesites.

In some embodiments, the present invention provides chirally controlled(and/or stereochemically pure) oligonucleotide compositions comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers. A pattern of        backbone chiral centers of an oligonucleotide can be designated        by a combination of linkage phosphorus stereochemistry (Rp/Sp)        from 5′ to 3′. For example, as exemplified below ONT-154 has a        pattern of 5S-(SSR)₃-5S, and ONT-80 has S₁₉.

In some embodiments, the present invention provides chirally controlledoligonucleotide composition of oligonucleotides in that the compositionis enriched, relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide type.In some embodiments, the present invention provides chirally controlledoligonucleotide composition of oligonucleotides in that the compositionis enriched, relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide typethat share:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers.

In some embodiments, in a substantially racemic (or chirallyuncontrolled) preparation of oligonucleotides, all or most couplingsteps are not chirally controlled in that the coupling steps are notspecifically conducted to provide enhanced stereoselectivity. Anexemplary substantially racemic preparation of oligonucleotides is thepreparation of phosphorothioate oligonucleotides through sulfurizingphosphite triesters with either tteraethylthiuram disulfide or (TETD) or3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD), a well-known process inthe art. In some embodiments, substantially racemic preparation ofoligonucleotides provides substantially racemic oligonucleotidecompositions (or chirally uncontrolled oligonucleotide compositions).

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type. In some        embodiments, a chirally controlled oligonucleotide composition        is a substantially pure preparation of a oligonucleotide type in        that oligonucleotides in the composition that are not of the        oligonucleotide type are impurities form the preparation process        of said oligonucleotide type, in some case, after certain        purification procedures.

In some embodiments, at least about 20% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 25% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 30% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 35% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 40% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 45% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 50% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 55% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 60% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 65% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 70% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 75% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 80% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 85% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 90% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 92% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 94% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 95% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% of the oligonucleotides in the composition have a common basesequence and length, a common pattern of backbone linkages, and a commonpattern of backbone chiral centers. In some embodiments, greater thanabout 99% of the oligonucleotides in the composition have a common basesequence and length, a common pattern of backbone linkages, and a commonpattern of backbone chiral centers. In some embodiments, purity of achirally controlled oligonucleotide composition of an oligonucleotidecan be expressed as the percentage of oligonucleotides in thecomposition that have a common base sequence and length, a commonpattern of backbone linkages, and a common pattern of backbone chiralcenters.

In some embodiments, purity of a chirally controlled oligonucleotidecomposition of an oligonucleotide type is expressed as the percentage ofoligonucleotides in the composition that are of the oligonucleotidetype. In some embodiments, at least about 10% of the oligonucleotides ina chirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 20% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 30% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 40% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 50% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 60% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 70% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 80% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 90% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 92% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 94% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 95% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 96% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 97% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 98% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 99% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type.

In some embodiments, purity of a chirally controlled oligonucleotidecomposition can be controlled by stereoselectivity of each coupling stepin its preparation process. In some embodiments, a coupling step has astereoselectivity (e.g., diastereoselectivity) of 60% (60% of the newinternucleotidic linkage formed from the coupling step has the intendedstereochemistry). After such a coupling step, the new internucleotidiclinkage formed may be referred to have a 60% purity. In someembodiments, each coupling step has a stereoselectivity of at least 60%.In some embodiments, each coupling step has a stereoselectivity of atleast 70%. In some embodiments, each coupling step has astereoselectivity of at least 80%. In some embodiments, each couplingstep has a stereoselectivity of at least 85%. In some embodiments, eachcoupling step has a stereoselectivity of at least 90%. In someembodiments, each coupling step has a stereoselectivity of at least 91%.In some embodiments, each coupling step has a stereoselectivity of atleast 92%. In some embodiments, each coupling step has astereoselectivity of at least 93%. In some embodiments, each couplingstep has a stereoselectivity of at least 94%. In some embodiments, eachcoupling step has a stereoselectivity of at least 95%. In someembodiments, each coupling step has a stereoselectivity of at least 96%.In some embodiments, each coupling step has a stereoselectivity of atleast 97%. In some embodiments, each coupling step has astereoselectivity of at least 98%. In some embodiments, each couplingstep has a stereoselectivity of at least 99%. In some embodiments, eachcoupling step has a stereoselectivity of at least 99.5%. In someembodiments, each coupling step has a stereoselectivity of virtually100%. In some embodiments, a coupling step has a stereoselectivity ofvirtually 100% in that all detectable product from the coupling step byan analytical method (e.g., NMR, HPLC, etc) has the intendedstereoselectivity.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are antisense oligonucleotides(e.g., chiromersen). In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are siRNA oligonucleotides.In some embodiments, a provided chirally controlled oligonucleotidecomposition is of oligonucleotides that can be antisenseoligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir,ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide,triplex forming oligonucleotide, aptamer or adjuvant. In someembodiments, a chirally controlled oligonucleotide composition is ofantisense oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of antagomir oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofmicroRNA oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of pre-microRNA oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofantimir oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of supermir oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofribozyme oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of U1 adaptor oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is of RNAactivator oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of RNAi agent oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofdecoy oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of triplex forming oligonucleotides. Insome embodiments, a chirally controlled oligonucleotide composition isof aptamer oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of adjuvant oligonucleotides.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides that includeone or more modified backbone linkages, bases, and/or sugars.

In some embodiments, a provided oligonucleotide comprises one or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide comprises two or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide comprisesthree or more chiral, modified phosphate linkages. In some embodiments,a provided oligonucleotide comprises four or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotidecomprises five or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25chiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 5 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises6 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 7 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 8 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 9 or more chiral,modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 10 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises11 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 12 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 13 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 14 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 15 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises16 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 17 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 18 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 19 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 20 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises21 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 22 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 23 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 24 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 25 or more chiral, modified phosphatelinkages.

In some embodiments, a provided oligonucleotide comprises at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% chiral, modified phosphate linkages.Exemplary such chiral, modified phosphate linkages are described aboveand herein. In some embodiments, a provided oligonucleotide comprises atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral, modified phosphatelinkages in the Sp configuration.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 80%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 85%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 90%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 91%.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 92%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 93%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 94%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 95%.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 96%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 97%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 98%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 99%.

In some embodiments, a chiral, modified phosphate linkage is a chiralphosphorothioate linkage, i.e., phosphorothioate internucleotidiclinkage. In some embodiments, a provided oligonucleotide comprises atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral phosphorothioateinternucleotidic linkages. In some embodiments, all chiral, modifiedphosphate linkages are chiral phosphorothioate internucleotidiclinkages. In some embodiments, at least about 10, 20, 30, 40, 50, 60,70, 80, or 90% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 10% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Sp conformation. Insome embodiments, at least about 20% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Spconformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Sp conformation. In some embodiments, at least about 40%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Sp conformation. In some embodiments, atleast about 50% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 60% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Sp conformation. Insome embodiments, at least about 70% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Spconformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Sp conformation. In some embodiments, at least about 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Sp conformation. In some embodiments, atleast about 95% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, atleast about 10% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, at least about 20% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Rp conformation. Insome embodiments, at least about 30% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, at least about 50%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, atleast about 60% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, at least about 70% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Rp conformation. Insome embodiments, at least about 80% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, at least about 95%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 10%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 20% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 30% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, less than about 40% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 50%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 60% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 70% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, less than about 80% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 95% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, a provided oligonucleotide has only one Rp chiralphosphorothioate internucleotidic linkages. In some embodiments, aprovided oligonucleotide has only one Rp chiral phosphorothioateinternucleotidic linkages, wherein all internucleotide linkages arechiral phosphorothioate internucleotidic linkages. In some embodiments,a chiral phosphorothioate internucleotidic linkage is a chiralphosphorothioate diester linkage. In some embodiments, each chiralphosphorothioate internucleotidic linkage is independently a chiralphosphorothioate diester linkage. In some embodiments, eachinternucleotidic linkage is independently a chiral phosphorothioatediester linkage. In some embodiments, each internucleotidic linkage isindependently a chiral phosphorothioate diester linkage, and only one isRp.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides that containone or more modified bases. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are ofoligonucleotides that contain no modified bases. Exemplary such modifiedbases are described above and herein.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 8 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 9 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 10 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 11 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 12bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 13 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 14 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 15 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 16 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 17bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 18 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 19 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 20 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 21 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 22bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 23 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 24 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 25 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 30, 35, 40, 45, 50, 55, 60,65, 70, or 75 bases.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations comprise oligonucleotides containingone or more residues which are modified at the sugar moiety. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations comprise oligonucleotides containing one or more residueswhich are modified at the 2′ position of the sugar moiety (referred toherein as a “2′-modification”). Exemplary such modifications aredescribed above and herein and include, but are not limited to, 2′-OMe,2′-MOE, 2′-LNA, 2′-F, etc. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations compriseoligonucleotides containing one or more residues which are 2′-modified.For example, in some embodiments, provided oligonucleotides contain oneor more residues which are 2′-O-methoxyethyl (2′-MOE)-modified residues.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations comprise oligonucleotides which donot contain any 2′-modifications. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations areoligonucleotides which do not contain any 2′-MOE residues. That is, insome embodiments, provided oligonucleotides are not MOE-modified.

In some embodiments, provided chirally controlled (and/orstereochemically pure) oligonucleotides are of a general motif ofwing-core-wing (also represented herein generally as X—Y—X). In someembodiments, each wing contains one or more residues having a particularmodification, which modification is absent from the core “Y” portion. Insome embodiment, each wing contains one or more residues having a 2′modification that is not present in the core portion. For instance, insome embodiments, provided chirally controlled (and/or stereochemicallypure) oligonucleotides have a wing-core-wing motif represented as X-Y-X,wherein the residues at each “X” portion are 2′-modified residues of aparticular type and the residues in the core “Y” portion are not2′-modified residues of the same particular type. For instance, in someembodiments, provided chirally controlled (and/or stereochemically pure)oligonucleotides have a wing-core-wing motif represented as X—Y—X,wherein the residues at each “X” portion are 2′-MOE-modified residuesand the residues in the core “Y” portion are not 2′-MOE-modifiedresidues. In some embodiments, provided chirally controlled (and/orstereochemically pure) oligonucleotides have a wing-core-wing motifrepresented as X—Y—X, wherein the residues at each “X” portion are2′-MOE-modified residues and the residues in the core “Y” portion are2′-deoxyribonucleotide. One of skill in the relevant arts will recognizethat all such 2′-modifications described above and herein arecontemplated in the context of such X—Y—X motifs.

In some embodiments, each wing region independently has a length of oneor more bases. In some embodiments, each wing region independently has alength of two or more bases. In some embodiments, each wing regionindependently has a length of three or more bases. In some embodiments,each wing region independently has a length of four or more bases. Insome embodiments, each wing region independently has a length of five ormore bases. In some embodiments, each wing region independently has alength of six or more bases. In some embodiments, each wing regionindependently has a length of seven or more bases. In some embodiments,each wing region independently has a length of eight or more bases. Insome embodiments, each wing region independently has a length of nine ormore bases. In some embodiments, each wing region independently has alength of ten or more bases. In certain embodiments, each wing regionhas a length of one base. In certain embodiments, each wing region has alength of two bases. In certain embodiments, each wing region has alength of three bases. In certain embodiments, each wing region has alength of four bases. In certain embodiments, each wing region has alength of five bases.

In some embodiments, a core region has a length of one or more bases. Insome embodiments, a core region has a length of one or more bases. Insome embodiments, a core region has a length of two or more bases. Insome embodiments, a core region has a length of three or more bases. Insome embodiments, a core region has a length of four or more bases. Insome embodiments, a core region has a length of five or more bases. Insome embodiments, a core region has a length of six or more bases. Insome embodiments, a core region has a length of seven or more bases. Insome embodiments, a core region has a length of eight or more bases. Insome embodiments, a core region has a length of nine or more bases. Insome embodiments, a core region has a length of ten or more bases. Insome embodiments, a core region has a length of 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, or more bases. In certain embodiments, a core regionhas a length of ten bases. In certain embodiments, a core region has alength of 3 bases. In certain embodiments, a core region has a length of4 bases. In certain embodiments, a core region has a length of 5 bases.In certain embodiments, a core region has a length of 6 bases. Incertain embodiments, a core region has a length of 7 bases. In certainembodiments, a core region has a length of 8 bases. In certainembodiments, a core region has a length of 9 bases. In certainembodiments, a core region has a length of 10 bases. In certainembodiments, a core region has a length of 11 bases. In certainembodiments, a core region has a length of 12 bases. In certainembodiments, a core region has a length of 13 bases. In certainembodiments, a core region has a length of 14 bases. In certainembodiments, a core region has a length of 15 bases. In certainembodiments, a core region has a length of 16 bases. In certainembodiments, a core region has a length of 17 bases. In certainembodiments, a core region has a length of 18 bases. In certainembodiments, a core region has a length of 19 bases. In certainembodiments, a core region has a length of 11 or more bases. In certainembodiments, a core region has a length of 12 or more bases. In certainembodiments, a core region has a length of 13 or more bases. In certainembodiments, a core region has a length of 14 or more bases. In certainembodiments, a core region has a length of 15 or more bases. In certainembodiments, a core region has a length of 16 or more bases. In certainembodiments, a core region has a length of 17 or more bases. In certainembodiments, a core region has a length of 18 or more bases. In certainembodiments, a core region has a length of 19 or more bases. In certainembodiments, a core region has a length of 20 or more bases. In certainembodiments, a core region has a length of more than 20 bases.

In some embodiments, a wing-core-wing (i.e., X—Y—X) motif of a providedoligonucleotide is represented numerically as, e.g., 5-10-5, meaningeach wing region of the oligonucleotide is 5 bases in length and thecore region of the oligonucleotide is 10 bases in length. In someembodiments, a wing-core-wing motif is any of, e.g. 2-16-2, 3-14-3,4-12-4, 5-10-5, etc. In certain embodiments, a wing-core-wing motif is5-10-5.

In some embodiments, the internucleosidic linkages of providedoligonucleotides of such wing-core-wing (i.e., X—Y—X) motifs are allchiral, modified phosphate linkages. In some embodiments, theinternucleosidic linkages of provided oligonucleotides of suchwing-core-wing (i.e., X—Y—X) motifs are all chiral phosphorothioateinternucleotidic linkages. In some embodiments, chiral internucleotidiclinkages of provided oligonucleotides of such wing-core-wing motifs areat least about 10, 20, 30, 40, 50, 50, 70, 80, or 90% chiral, modifiedphosphate internucleotidic linkages. In some embodiments, chiralinternucleotidic linkages of provided oligonucleotides of suchwing-core-wing motifs are at least about 10, 20, 30, 40, 50, 60, 70, 80,or 90% chiral phosphorothioate internucleotidic linkages. In someembodiments, chiral internucleotidic linkages of providedoligonucleotides of such wing-core-wing motifs are at least about 10,20, 30, 40, 50, 50, 70, 80, or 90% chiral phosphorothioateinternucleotidic linkages of the Sp conformation.

In some embodiments, each wing region of a wing-core-wing motifoptionally contains chiral, modified phosphate internucleotidiclinkages. In some embodiments, each wing region of a wing-core-wingmotif optionally contains chiral phosphorothioate internucleotidiclinkages. In some embodiments, each wing region of a wing-core-wingmotif contains chiral phosphorothioate internucleotidic linkages. Insome embodiments, the two wing regions of a wing-core-wing motif havethe same internucleotidic linkage stereochemistry. In some embodiments,the two wing regions have different internucleotidic linkagestereochemistry. In some embodiments, each internucleotidic linkage inthe wings is independently a chiral internucleotidic linkage.

In some embodiments, the core region of a wing-core-wing motifoptionally contains chiral, modified phosphate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifoptionally contains chiral phosphorothioate internucleotidic linkages.In some embodiments, the core region of a wing-core-wing motif comprisesa repeating pattern of internucleotidic linkage stereochemistry. In someembodiments, the core region of a wing-core-wing motif has a repeatingpattern of internucleotidic linkage stereochemistry. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)mRp or Rp(Sp)m, wherein m is 2, 3, 4, 5, 6,7 or 8. In some embodiments, the core region of a wing-core-wing motifhas repeating pattern of internucleotidic linkage stereochemistry,wherein the repeating pattern is (Sp)mRp or Rp(Sp)m, wherein m is 2, 3,4, 5, 6, 7 or 8. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is (Sp)mRp, wherein m is2, 3, 4, 5, 6, 7 or 8. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is Rp(Sp)m, wherein ni is2, 3, 4, 5, 6, 7 or 8. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is (Sp)mRp or Rp(Sp)m,wherein m is 2, 3, 4, 5, 6, 7 or 8, In some embodiments, the core regionof a wing-core-wing motif has repeating pattern of internucleotidiclinkage stereochemistry, wherein the repeating pattern is a motifcomprising at least 33% of internucleotidic linkage in the Sconformation. In some embodiments, the core region of a wing-core-wingmotif has repeating pattern of internucleotidic linkage stereochemistry,wherein the repeating pattern is a motif comprising at least 50% ofinternucleotidic linkage in the S conformation. In some embodiments, thecore region of a wing-core-wing motif has repeating pattern ofinternucleotidic linkage stereochemistry, wherein the repeating patternis a motif comprising at least 66% of internucleotidic linkage in the Sconformation. In some embodiments, the core region of a wing-core-wingmotif has repeating pattern of internucleotidic linkage stereochemistry,wherein the repeating pattern is a repeating triplet motif selected fromRpRpSp and SpSpRp. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is a repeating RpRpSp. Insome embodiments, the core region of a wing-core-wing motif hasrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is a repeating SPSpRp.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers in the core region comprises (Sp)mRpor Rp(Sp)m. In some embodiments, the present invention provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers in the core regioncomprises Rp(Sp)m. In some embodiments, the present invention provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers in the core regioncomprises (Sp)mRp. In some embodiments, m is 2. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises Rp(Sp)₂. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Sp)₂Rp(Sp)₂. In some embodiments,the present invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Rp)₂Rp(Sp)₂. In some embodiments,the present invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises RpSpRp(Sp)₂. In some embodiments,the present invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises SpRpRp(Sp)₂. In some embodiments,the present invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Sp)₂Rp.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)mRp or Rp(Sp)m. Insonic embodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises Rp(Sp)m. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters comprises (Sp)mRp. In some embodiments, m is 2. In someembodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises Rp(Sp)₂. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters comprises (Sp)₂Rp(Sp)₂. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises (Rp)₂Rp(SP)₂. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers comprisesRpSpRp(Sp)₂. In some embodiments, the present invention provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers comprises SpRpRp(Sp)₂. Insome embodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Sp)₂Rp.

As defined herein, m is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, m is3, 4, 5, 6, 7 or 8. In some embodiments, m is 4, 5, 6, 7 or 8. In someembodiments, M is 5, 6, 7 or 8. In some embodiments, m is 6, 7 or 8. Insome embodiments, m is 7 or 8. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments, mis 5. In some embodiments, m is 6. In some embodiments, m is 7. In someembodiments, m is 8.

In some embodiments, a repeating pattern is (Sp)m(Rp)n, wherein n isindependently 1, 2, 3, 4, 5, 6, 7 or 8, and m is independently asdefined above and described herein. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises (Sp)m(Rp)n. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Sp)m(Rp)n. In some embodiments, a repeatingpattern is (Rp)n(Sp)m, wherein n is independently 1, 2, 3, 4, 5, 6, 7 or8, and m is independently as defined above and described herein. In someembodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Rp)n(Sp)m. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Rp)n(Sp)m. In some embodiments,(Rp)n(Sp)m is (Rp)(Sp)₂. In some embodiments, (Sp)n(Rp)m is (Sp)₂(Rp).

In some embodiments, a repeating pattern is (Sp)m(Rp)n(Sp)t, whereineach of n and t is independently 1, 2, 3, 4, 5, 6, 7 or 8, and m is asdefined above and described herein. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises (Sp)m(Rp)n(Sp)t. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Sp)m(Rp)n(Sp)t. In some embodiments, a repeatingpattern is (Sp)t(Rp)n(Sp)m, wherein each of n and t is independently 1,2, 3, 4, 5, 6, 7 or 8, and m is as defined above and described herein.In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)t(Rp)n(Sp)m. In someembodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Sp)t(Rp)n(Sp)m.

In some embodiments, a repeating pattern is (Np)t(Rp)n(Sp)m, whereineach of n and t is independently 1, 2, 3, 4, 5, 6, 7 or 8, Np isindependently Rp or Sp, and m is as defined above and described herein.In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Np)t(Rp)n(Sp)m. In someembodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Np)t(Rp)n(Sp)m. Insome embodiments, a repeating pattern is (Np)m(Rp)n(Sp)t, wherein eachof n and t is independently 1, 2, 3, 4, 5, 6, 7 or 8, Np isindependently Rp or Sp, and m is as defined above and described herein.In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Np)m(Rp)n(Sp)t. In someembodiments, the present invention provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Np)m(Rp)n(Sp)t. Insome embodiments, Np is Rp. In some embodiments, Np is Sp. In someembodiments, all Np are the same. In some embodiments, all Np are Sp. Insome embodiments, at least one Np is different from the other Np. Insome embodiments, t is 2.

As defined herein, n is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, nis 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 3, 4, 5, 6, 7 or 8.In some embodiments, n is 4, 5, 6, 7 or 8. In some embodiments, n is 5,6, 7 or 8. In some embodiments, n is 6, 7 or 8. In some embodiments, nis 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2. Insome embodiments, n is 3. In some embodiments, n is 4. In someembodiments, n is 5. In some embodiments, n is 6. In some embodiments, nis 7. In some embodiments, n is 8.

As defined herein, t is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, tis 2, 3, 4, 5, 6, 7 or 8. In some embodiments, t is 3, 4, 5, 6, 7 or 8.In some embodiments, t is 4, 5, 6, 7 or 8. In some embodiments, t is 5,6, 7 or 8. In some embodiments, t is 6, 7 or 8. In sonic embodiments, tis 7 or 8. In some embodiments, t is 1. In some embodiments, t is 2. Insome embodiments, t is 3. In some embodiments, t is 4. In someembodiments, t is 5. In some embodiments, t is 6. in some embodiments, tis 7. In some embodiments, t is 8.

In some embodiments, at least one of m and t is greater than 2. In someembodiments, at least one of m and tis greater than 3. In someembodiments, at least one of m and t is greater than 4. In someembodiments, at least one of m and t is greater than 5. In someembodiments, at least one of m and t is greater than 6. In someembodiments, at least one of m and t is greater than 7. In someembodiments, each one of m and t is greater than 2. In some embodiments,each one of m and t is greater than 3. In some embodiments, each one ofm and t is greater than 4. In some embodiments, each one of m and t isgreater than 5. In some embodiments, each one of m and t is greater than6. In some embodiments, each one of m and t is greater than 7.

In some embodiments, n is 1, and at least one of m and t is greaterthan 1. In some embodiments, n is 1 and each of m and t is independentgreater than 1. In some embodiments, m>n and t>n. In some embodiments,(Sp)m(Rp)n(Sp)t is (Sp)₂Rp(Sp)₂. In some embodiments, (Sp)t(Rp)n(Sp)m is(Sp)₂Rp(Sp)₂. In some embodiments, (Sp)t(Rp)n(Sp)m is SpRp(Sp)₂. In someembodiments, (Np)t(Rp)n(Sp)m is (Np)tRp(Sp)m. In some embodiments,(Np)t(Rp)n(Sp)m is (Np)₂Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is(Rp)₂Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is (Sp)₂Rp(Sp)m. Insome embodiments, (Np)t(Rp)n(Sp)m is RpSpRp(Sp)m. In some embodiments,(Np)t(Rp)n(Sp)m is SpRpRp(Sp)m.

In some embodiments, (Sp)t(Rp)n(Sp)m is SpRpSpSp. In some embodiments,(Sp)t(Rp)n(Sp)m is (Sp)₂Rp(Sp)₂. In some embodiments, (Sp)t(Rp)n(Sp)m is(Sp)₃Rp(Sp)₃. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)₄Rp(Sp)₄. Insome embodiments, (Sp)t(Rp)n(Sp)m is (Sp)tRp(Sp)₅. In some embodiments,(Sp)t(Rp)n(Sp)m is SpRp(Sp)₅. In some embodiments, (Sp)t(Rp)n(Sp)m is(Sp)₂Rp(Sp)₅. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)₃Rp(Sp)₅. Insome embodiments, (Sp)t(Rp)n(Sp)m is (Sp)₄Rp(Sp)₅. In some embodiments,(Sp)t(Rp)n(Sp)m is (Sp)5Rp(Sp)₅.

In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)₂Rp(Sp)₂. In someembodiments, (Sp)m(Rp)n(Sp)t is (Sp)₃Rp(Sp)₃. In some embodiments,(Sp)m(Rp)n(Sp)t is (Sp)₄Rp(Sp)₄. In some embodiments, (Sp)m(Rp)n(Sp)t is(Sp)mRp(Sp)₅. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)₂Rp(Sp)₅. Insome embodiments, (Sp)m(Rp)n(Sp)t is (Sp)₃Rp(Sp)₅. In some embodiments,(Sp)m(Rp)n(Sp)t is (Sp)₄Rp(Sp)₅. In some embodiments, (Sp)m(Rp)n(Sp)t is(Sp)₅Rp(Sp)₅.

In some embodiments, the core region of a wing-core-wing motif comprisesat least one Rp internucleotidic linkage. In some embodiments, the coreregion of a wing-core-wing motif comprises at least one Rpphosphorothioate internucleotidic linkage. In some embodiments, the coreregion of a wing-core-wing motif comprises at least two Rpinternucleotidic linkages. In some embodiments, the core region of awing-core-wing motif comprises at least two Rp phosphorothioateinternucleotidic linkages. In some embodiments, the core region of awing-core-wing motif comprises at least three Rp internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifcomprises at least three Rp phosphorothioate internucleotidic linkages.In some embodiments, the core region of a wing-core-wing motif comprisesat least 4, 5, 6, 7, 8, 9, or 10 Rp internucleotidic linkages. In someembodiments, the core region of a wing-core-wing motif comprises atleast 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioate internucleotidiclinkages.

In certain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues at each “X” wing region are 2′-MOE-modified residues. Incertain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues in the core “Y” region are 2′-deoxyribonucleotide residues.In certain embodiments, a wing-core-wing motif is a 5-10-5 motif,wherein all internucleosidic linkages are phosphorothioateinternucleosidic linkages. In certain embodiments, a wing-core-wingmotif is a 5-10-5 motif, wherein all internucleosidic linkages arechiral phosphorothioate internucleosidic linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each “X” wing region are 2′-MOE-modified residues, theresidues in the core “Y” region are 2′-deoxyribonucleotide, and allinternucleosidic linkages are chiral phosphorothioate internucleosidiclinkages.

In certain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues at each “X” wing region are not 2′-MOE-modified residues.In certain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues in the core “Y” region are 2′-deoxyribonucleotide residues.In certain embodiments, a wing-core-wing motif is a 5-10-5 motif,wherein all internucleosidic linkages are phosphorothioateinternucleosidic linkages. In certain embodiments, a wing-core-wingmotif is a 5-10-5 motif, wherein all internucleosidic linkages arechiral phosphorothioate internucleosidic linkages. In certainembodiments, a wing- core-wing motif is a 5-10-5 motif wherein theresidues at each “X” wing region are not 2′-MOE-modified residues, theresidues in the core “Y” region are 2′-deoxyribonucleotide, and allinternucleosidic linkages are chiral phosphorothioate internucleosidiclinkages.

In certain embodiments, provided chirally controlled (and/orstereochemically pure) preparations comprise oligonucleotides of basesequence GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9).

In some embodiments, the present invention provides stereochemicaldesign parameters for oligonucleotides. That is, among other things, thepresent disclosure demonstrates impact of stereochemical structure atdifferent positions along an oligonucleotide chain, for example onstability and/or activity of the oligonucleotide, including oninteraction of the oligonucleotide with a cognate ligand and/or with aprocessing enzyme. The present invention specifically providesoligonucleotides whose structure incorporates or reflects the designparameters. Such oligonucleotides are new chemical entities relative tostereorandom preparations having the same base sequence and length.

In some embodiments, the present invention provides stereochemicaldesign parameters for antisense oligonucleotides. In some embodiments,the present invention specifically provides design parameter foroligonucleotides that may be bound and/or cleaved by RNaseH. In omeembodiments, the present invention provides stereochemical designparameters for siRNA oligonucleotides. In some embodiments, the presentinvention specifically provides design parameters for oligonucleotidesthat may be bound and/or cleaved by, e.g., DICER, Argonaute proteins(e.g., Argonaute-1 and Argonaute-2), etc.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages is chiral. In some embodiments, at least twoof the first, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, at least three of the first, second, third, fifth, seventh,eighth, ninth, eighteenth, nineteenth and twentieth internucleotidiclinkages are chiral. In some embodiments, at least four of the first,second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth andtwentieth internucleotidic linkages are chiral. In some embodiments, atleast five of the first, second, third, fifth, seventh, eighth, ninth,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral. In some embodiments, at least six of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at leastseven of the first, second, third, fifth, seventh, eighth, ninth,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral. In some embodiments, at least eight of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at least nineof the first, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, one of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkages ischiral. In some embodiments, two of the first, second, third, fifth,seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, three of thefirst, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, four of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral. In some embodiments, five of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, six of thefirst, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, seven of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral. In some embodiments, eight of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, nine of thefirst, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, ten of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighteenth, nineteenth and twentieth internucleotidiclinkages is chiral. In some embodiments, at least two of the first,second, third, fifth, seventh, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at leastthree of the first, second, third, fifth, seventh, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, at least four of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral. In some embodiments, at least five of the first, second, third,fifth, seventh, eighteenth, nineteenth and twentieth internucleotidiclinkages are chiral. In some embodiments, at least six of the first,second, third, fifth, seventh, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at leastseven of the first, second, third, fifth, seventh, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, one of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages ischiral. In some embodiments, two of the first, second, third, fifth,seventh, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral. In some embodiments, three of the first, second, third,fifth, seventh, eighteenth, nineteenth and twentieth internucleotidiclinkages are chiral. In some embodiments, four of the first, second,third, fifth, seventh, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, five of thefirst, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages are chiral. In some embodiments, sixof the first, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages are chiral. In some embodiments,seven of the first, second, third, fifth, seventh, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, eight of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages is chiral, and at least one internucleotidiclinkage is achiral. In some embodiments, a single oligonucleotide of aprovided composition comprises a region in which at least one of thefirst, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages is chiral, and at least oneinternucleotidic linkage is achiral. In some embodiments, at least twointernucleotidic linkages are achiral. In some embodiments, at leastthree internucleotidic linkages are achiral. In some embodiments, atleast four internucleotidic linkages are achiral. In some embodiments,at least five internucleotidic linkages are achiral. In someembodiments, at least six internucleotidic linkages are achiral. In someembodiments, at least seven internucleotidic linkages are achiral. Insome embodiments, at least eight internucleotidic linkages are achiral.In some embodiments, at least nine internucleotidic linkages areachiral. In some embodiments, at least 10 internucleotidic linkages areachiral. In some embodiments, at least 11 internucleotidic linkages areachiral. In some embodiments, at least 12 internucleotidic linkages areachiral. In some embodiments, at least 13 internucleotidic linkages areachiral. In some embodiments, at least 14 internucleotidic linkages areachiral. In some embodiments, at least 15 internucleotidic linkages areachiral. In some embodiments, at least 16 internucleotidic linkages areachiral. In some embodiments, at least 17 internucleotidic linkages areachiral. In some embodiments, at least 18 internucleotidic linkages areachiral. In some embodiments, at least 19 internucleotidic linkages areachiral. In some embodiments, at least 20 internucleotidic linkages areachiral. In some embodiments, one internucleotidic linkage is achiral.In some embodiments, two internucleotidic linkages are achiral. In someembodiments, three internucleotidic linkages are achiral. In someembodiments, four internucleotidic linkages are achiral. In someembodiments, five internucleotidic linkages are achiral. In someembodiments, six internucleotidic linkages are achiral. In someembodiments, seven internucleotidic linkages are achiral. In someembodiments, eight internucleotidic linkages are achiral. In someembodiments, nine internucleotidic linkages are achiral. In someembodiments, 10 internucleotidic linkages are achiral. In someembodiments, 11 internucleotidic linkages are achiral. In someembodiments, 12 internucleotidic linkages are achiral. In someembodiments, 13 internucleotidic linkages are achiral. In someembodiments, 14 internucleotidic linkages are achiral. In someembodiments, 15 internucleotidic linkages are achiral. In someembodiments, 16 internucleotidic linkages are achiral. In someembodiments, 17 internucleotidic linkages are achiral. In someembodiments, 18 internucleotidic linkages are achiral. In someembodiments, 19 internucleotidic linkages are achiral. In someembodiments, 20 internucleotidic linkages are achiral. In someembodiments, a single oligonucleotide of a provided compositioncomprises a region in which all internucleotidic linkages, except the atleast one of the first, second, third, fifth, seventh, eighth, ninth,eighteenth, nineteenth and twentieth internucleotidic linkages which ischiral, are achiral.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages is chiral, and at least one internucleotidiclinkage is phosphate. In some embodiments, a single oligonucleotide of aprovided composition comprises a region in which at least one of thefirst, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages is chiral, and at least oneinternucleotidic linkage is phosphate. In some embodiments, at least twointernucleotidic linkages are phosphate. In some embodiments, at leastthree internucleotidic linkages are phosphate. In some embodiments, atleast four internucleotidic linkages are phosphate. In some embodiments,at least five internucleotidic linkages are phosphate. In someembodiments, at least six internucleotidic linkages are phosphate. Insome embodiments, at least seven internucleotidic linkages arephosphate. In some embodiments, at least eight internucleotidic linkagesare phosphate. In some embodiments, at least nine internucleotidiclinkages are phosphate. In some embodiments, at least 10internucleotidic linkages are phosphate. In some embodiments, at least11 internucleotidic linkages are phosphate. In some embodiments, atleast 12 internucleotidic linkages are phosphate. In some embodiments,at least 13 internucleotidic linkages are phosphate. In someembodiments, at least 14 internucleotidic linkages are phosphate. Insome embodiments, at least 15 internucleotidic linkages are phosphate.In some embodiments, at least 16 internucleotidic linkages arephosphate. In some embodiments, at least 17 internucleotidic linkagesare phosphate. In some embodiments, at least 18 internucleotidiclinkages are phosphate. In some embodiments, at least 19internucleotidic linkages are phosphate. In some embodiments, at least20 internucleotidic linkages are phosphate. In some embodiments, oneinternucleotidic linkage is phosphate. In some embodiments, twointernucleotidic linkages are phosphate. In some embodiments, threeinternucleotidic linkages are phosphate. In some embodiments, fourinternucleotidic linkages are phosphate. In some embodiments, fiveinternucleotidic linkages are phosphate. In some embodiments, sixinternucleotidic linkages are phosphate. In some embodiments, seveninternucleotidic linkages are phosphate. In some embodiments, eightinternucleotidic linkages are phosphate. In some embodiments, nineinternucleotidic linkages are phosphate. In some embodiments, 10internucleotidic linkages are phosphate. In some embodiments, 11internucleotidic linkages are phosphate. In some embodiments, 12internucleotidic linkages are phosphate. In some embodiments, 13internucleotidic linkages are phosphate. In some embodiments, 14internucleotidic linkages are phosphate. In some embodiments, 15internucleotidic linkages are phosphate. In some embodiments, 16internucleotidic linkages are phosphate. In some embodiments, 17internucleotidic linkages are phosphate. In some embodiments, 18internucleotidic linkages are phosphate. In some embodiments, 19internucleotidic linkages are phosphate. In some embodiments, 20internucleotidic linkages are phosphate. In some embodiments, a singleoligonucleotide of a provided composition comprises a region in whichall internucleotidic linkages, except the at least one of the first,second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth andtwentieth internucleotidic linkages which is chiral, are phosphate.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral, and at least 10% of all theinternucleotidic linkages in the region is achiral. In some embodiments,a single oligonucleotide of a provided composition comprises a region inwhich at least one of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages ischiral, and at least 10% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 20% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 30% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 40% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 50% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 60% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 70% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 80% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 90% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 50% of all theinternucleotidic linkages in the region are achiral. In someembodiments, an achiral internucleotidic linkage is a phosphate linkage.In some embodiments, each achiral internucleotidic linkage in aphosphate linkage.

In some embodiments, the first internucleotidic linkage of the region isan Sp modified internucleotidic linkage. In some embodiments, the firstinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the secondinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the secondinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the thirdinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the thirdinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the fifthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the fifthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the seventhinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the seventhinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the eighthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the eighthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the ninthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the ninthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the eighteenthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the eighteenthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the nineteenthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the nineteenthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the twentiethinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the twentiethinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage.

In some embodiments, the region has a length of at least 21 bases. Insome embodiments, the region has a length of 21 bases. In someembodiments, a single oligonucleotide in a provided composition has alength of at least 21 bases. In some embodiments, a singleoligonucleotide in a provided composition has a length of 21 bases.

In some embodiments, a chiral internucleotidic linkage has the structureof formula I. In some embodiments, a chiral internucleotidic linkage isphosphorothioate. In some embodiments, each chiral internucleotidiclinkage in a single oligonucleotide of a provided compositionindependently has the structure of formula I. In some embodiments, eachchiral internucleotidic linkage in a single oligonucleotide of aprovided composition is a phosphorothioate.

As known by a person of ordinary skill in the art and described in thedisclosure, various modifications can be introduced to the 2′-positionof the sugar moiety. Commonly used 2′-modifications include but are notlimited to 2′-OR¹, wherein R¹ is not hydrogen. In some embodiments, amodification is 2′-OR, wherein R is optionally substituted aliphatic. Insome embodiments, a modification is 2′-OMe. In some embodiments, amodification is 2′-O—MOE. In some embodiments, the present inventiondemonstrates that inclusion and/or location of particular chirally pureinternucleotidic linkages can provide stability improvements comparableto or better than those achieved through use of modified backbonelinkages, bases, and/or sugars. In some embodiments, a provided singleoligonucleotide of a provided composition has no modifications on thesugars. In some embodiments, a provided single oligonucleotide of aprovided composition has no modifications on 2′-positions of the sugars(i.e., the two groups at the 2′-position are either —H/—H or —H/—OH). Insome embodiments, a provided single oligonucleotide of a providedcomposition does not have any 2′-MOE modifications.

In some embodiments, a single oligonucleotide in a provided compositionis a better substrate for Argonaute proteins (e.g., hAgo-1 and hAgo-2)compared to stereorandom oligonucleotide compositions. Selection and/orlocation of chirally pure linkages as described in the present closureare useful design parameters for oligonucleotides that interacting withsuch proteins, such as siRNA.

In some embodiments, a single oligonucleotide in a provided compositionhas at least about 25% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 30% of its internucleotidiclinkages in Sp configuration. In some embodiments, a singleoligonucleotide in a provided composition has at least about 35% of itsinternucleotidic linkages in Sp configuration. In some embodiments, asingle oligonucleotide in a provided composition has at least about 40%of its internucleotidic linkages in Sp configuration. In someembodiments, a single oligonucleotide in a provided composition has atleast about 45% of its internucleotidic linkages in Sp configuration. Insome embodiments, a single oligonucleotide in a provided composition hasat least about 50% of its internucleotidic linkages in Sp configuration.In some embodiments, a single oligonucleotide in a provided compositionhas at least about 55% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 60% of its internucleotidiclinkages in Sp configuration. In some embodiments, a singleoligonucleotide in a provided composition has at least about 65% of itsinternucleotidic linkages in Sp configuration. In some embodiments, asingle oligonucleotide in a provided composition has at least about 70%of its internucleotidic linkages in Sp configuration. In someembodiments, a single oligonucleotide in a provided composition has atleast about 75% of its internucleotidic linkages in Sp configuration. Insome embodiments, a single oligonucleotide in a provided composition hasat least about 80% of its internucleotidic linkages in Sp configuration.In some embodiments, a single oligonucleotide in a provided compositionhas at least about 85% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 90% of its internucleotidiclinkages in Sp configuration.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from:

143 (Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,  (SSR)₃- (SEQ IDRp, Sp, Sp)-d[5mCs1As1Gs1Ts15mCs1 SS NO: 10) Ts1Gs15mCs1Ts1Ts15mCs1G]ONT- (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, (5R- (SEQ ID 87Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp,   (SSR)₃- NO: 11)Rp, Rp, Rp)-Gs5mCs5mCsTs5mCsAsGsTs 5R)5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mC underlined nuecleotides are2′-modified.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from:

143 (Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,  (SSR)₃-SS (SEQ IDRp, Sp, Sp)-d[5mCs1As1Gs1Ts15mCs1T NO: 12) s1Gs15mCs1Ts1Ts15mCs1G] ONT-(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp,  (5R- (SEQ ID 87Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp,  (SSR)₃-5R) NO: 13)Rp, Rp, Rp)-Gs5mCs5mCsTs5mCsAsGsT s5mCsTsGs5mCsTsTs5mCsGs5mCsAs 5mCs5mCunderlined nuecleotides are 2′-O-MOE modified.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from:

ONT-106 (Rp)-uucuAGAccuGuuuuGcuudTsdT PCSK9 sense (SEQ ID NO: 14)ONT-107 (Sp)-uucuAGAccuGuuuuGcuudTsdT PCSK9 sense (SEQ ID NO: 15)ONT-108 (Rp)-AAGcAAAAcAGGUCuAGAAdTsdT PCSK9 antisense (SEQ ID NO: 16)ONT-109 (Sp)-AAGcAAAAcAGGUCuAGAAdTsdT PCSK9 antisense (SEQ ID NO: 17)ONT-110 (Rp, Rp)- PCSK9 antisense (SEQ ID asAGcAAAAcAGGUCuAGAAdTsdTNO: 18) ONT-111 (Sp, Rp)- PCSK9 antisense (SEQ IDasGcAAAAcAGGUCuAGAAdTsdT NO: 19) ONT-112 (Sp, Sp)- PCSK9 antisense(SEQ ID asGcAAAAcAGGUCuAGAAdTsdT NO: 20) ONT-113 (Rp, Sp)-PCSK9 antisense (SEQ ID asGcAAAAcAGGUCuAGAAdTsdT NO: 21) wherein lowercase letters represent 2'-OMe RNA residues; capital letters represent2'-OH RNA residues; and bolded and “s” indicates a phosphorothioatemoiety;and

PCSK9 (All (Sp))-ususcsusAsGsAscscsusGsus  (SEQ ID (1)usususGscsususdTsdT NO: 22) PCSK9 (All (Rp))-ususcsusAsGsAscscsusGsus(SEQ ID (2) usususGscsususdTsdT NO: 23) PCSK9(All (Sp))-usucuAsGsAsccuGsuuuuGscu (SEQ ID (3) usdTsdT NO: 24) PCSK9(All (Rp))-usucuAsGsAsccuGsuuuuGscu (SEQ ID (4) usdTsdT NO: 25) PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,  (SEQ ID (5)Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,  NO: 26)Sp, Rp, Sp)-ususcsusAsGsAscscsusGsu susususGscsususdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,  (SEQ ID (6)Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, NO: 27)Rp, Sp, Rp)-ususcsusAsGsAscscsusGsu susususGscsususdTsdT wherein lowercase letters represent 2′-OMe RNA residues; capital letters representRNA residues; d =2′-deoxy residues; and “s” indicates a phosphorothioatemoiety;and

PCSK9 (All (Rp))-AsAsGscsAsAsAsAscsAsGsGsU (SEQ ID (7)sCsusAsGsAsAsdTsdT NO: 28) PCSK9 (All (Sp))-AsAsGscsAsAsAsAscsAsGsGsU(SEQ ID (8) sCsusAsGsAsAsdTsdT NO: 29) PCSK9(All (Rp))-AsAGcAAAAcsAsGsGsUsCsusAs (SEQ ID (9) GsAsAsdTsdT NO: 30)PCSK9 (All (Sp))-AsAGcAAAAcsAsGsGsUsCsusAs (SEQ ID (10) GsAsAsdTsdTNO: 31) PCSK9 (All (Rp))-AAsGscsAsAsAsAscAGGUCuAGA (SEQ ID (11) AdTsdTNO: 32) PCSK9 (All (Sp))-AAsGscsAsAsAsAscAGGUCuAGA (SEQ ID (12) AdTsdTNO: 33) PCSK9 (All (Rp))-AsAsGscAsAsAsAscAsGsGsUsC (SEQ ID (13)suAsGsAsAsdTsdT NO: 34) PCSK9 (All (Sp))-AsAsGscAsAsAsAscAsGsGsUsC(SEQ ID (14) suAsGsAsAsdTsdT NO: 35) PCSK9(All (Rp))-AsAGcAAAsAscAsGsGsUsCsusA (SEQ ID (15) sGsAsAsdTsdT NO: 36)PCSK9 (All (Sp))-AsAGcAAAsAscAsGsGsUsCsusA (SEQ ID (16) sGsAsAsdTsdTNO: 37) PCSK9 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,  (SEQ ID (17)Sp, Rp, Sp, Rp, Sp)-AsAGcAAAsAscAsGs NO: 38) GsUsCsusAsGsAsAsdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,  (SEQ ID (18)Rp, Sp, Rp, Sp, Rp)-AsAGcAAAsAscAsGs NO: 39) GsUsCsusAsGsAsAsdTsdTwherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d = 2′-deoxy residues; “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Rp))-UfsusCfsusAfsgsAfscsCfsus (SEQ ID (19)GfsusUfsusUfsgsCfsusUfsdTsdT NO: 40) PCSK9(All (Sp))-UfsusCfsusAfsgsAfscsCfsus (SEQ ID (20)GfsusUfsusUfsgsCfsusUfsdTsdT NO: 41) PCSK9(All (Rp))-UfsuCfsuAfsgAfscCfsuGfsuU (SEQ ID (21) fsuUfsgCfsuUfsdTsdTNO: 42) PCSK9 (All (Sp))-UfsuCfsuAfsgAfscCfsuGfsuU  (SEQ ID (22)fsuUfsgCfsuUfsdTsdT NO: 43) PCSK9 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,(SEQ ID (23) Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, NO: 44)Rp, Sp)-UfsusCfsusAfsgsAfscsCfsusGfs usUfsusUfsgsCfsusUfsdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (24)Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, NO: 45)Sp, Rp)-UfsusCfsusAfsgsAfscsCfsusGfs usUfsusUfsgsCfsusUfsdTsdT whereinlower case letters represent 2′-OMe RNA residues; capital lettersrepresent 2′-F RNA residues; d = 2′-deoxy residues; and “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Rp))-asAfsgsCfsasAfsasAfscsAf (SEQ ID (25)sgsGfsusCfsusAfsgsAfsasdTsdT NO: 46) PCSK9(All (Sp))-asAfsgsCfsasAfsasAfscsAf (SEQ ID (26)sgsGfsusCfsusAfsgsAfsasdTsdT NO: 47) PCSK9(All (Rp))-asAfgCfaAfaAfcsAfsgsGfsu (SEQ ID (27) sCfsusAfsgsAfsasdTsdTNO: 48) PCSK9 (All (Sp))-asAfgCfaAfaAfcsAfsgsGfsu (SEQ ID (28)sCfsusAfsgsAfsasdTsdT NO: 49) PCSK9 (All (Rp))-asAfsgCfsaAfsaAfscAfsgGf(SEQ ID (29) suCfsuAfsgAfsadTsdT NO: 50) PCSK9(All (Sp))-asAfsgCfsaAfsaAfscAfsgGf (SEQ ID (30) suCfsuAfsgAfsadTsdTNO: 51) PCSK9 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (31)Sp, Rp, Sp, Rp, Sp)-asAfgCfaAfasAfs NO: 52)cAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (32)Rp, Sp, Rp, Sp, Rp)-asAfgCfaAfasAfs NO: 53)cAfsgsGfsusCfsusAfsgsAfsasdTsdT wherein lower case letters represent2′-OMe RNA residues; capital letters represent 2′-F RNA residues; d =2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from:d[A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C] (SEQ ID NO: 54),d[C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C_(S)C_(S)C] (SEQ ID NO: 55),d[C_(S)C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C] (SEQ ID NO: 56) andd[C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C] (SEQ ID NO: 57),wherein R is Rp phosphorothioate linkage, and S is Sp phosphorothioatelinkage.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from: GGA_(R)T_(S)G_(R)T_(S)T_(R)^(m)C_(S)TCGA (SEQ ID NO: 58), GGA_(R)T_(R)G_(S)T_(S)T_(R) ^(m)C_(R)TCGA(SEQ ID NO: 59), GGA_(S)T_(S)G_(R)T_(R)T_(S) ^(m)C_(S)TCGA (SEQ ID NO:60), wherein R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, all other linkages are PO, and each ^(m)C is a 5-methylcytosine modified nucleoside.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from : T_(k)T_(k)^(m)C_(k)AGT^(m)CATGA^(m)CT_(k)T^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 61),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methyl cytosine modified nucleoside, and allinternucleoside linkages are phosphorothioates (PS) with stereochemistrypatterns selected from RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR,RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS,RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRS SRSRS,S SRRRRRSRR, RSRRSRSS SR, RRS SRSRRRR, RRSRSRRS S S, RRSRSS SRRR,RSRRRRSRSR, S SRS SSRRRS, RS SRSRSRSR, RSRSRSSRS S, RRRS SRRSRS,SRRSSRRSRS, RRRRSRSRRR, SS S SRRRRSR, RRRRRRRRRR and SSSSSSSSSS.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from : T_(k)T_(k)^(m)C_(k)AGT^(m)CATGA^(m)CTT_(k) ^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 62),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methyl cytosine modified nucleoside and allinternucleoside linkages are phosphorothioates (PS) with stereochemistrypatterns selected from: RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR,RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSS SRRSS, RRRS SRRRSR, RRRRS S S SRS, SRRSRRRRRR, RS SRS SRRRR, RSRRSRRSRR,RRSRS SRSRS, S SRRRRRSRR, RSRRSRSS SR, RRS SRSRRRR, RRSRSRRS S S, RRSRSSSRRR, RSRRRRSRSR, S SRS SSRRRS, RS SRSRSRSR, RSRSRSSRS S, RRRS SRRSRS,SRRSSRRSRS, RRRRSRSRRR, SS S SRRRRSR, RRRRRRRRRR and SSSSSSSSSS.Modified Oligonucleotide Structures

As noted above, in light of the usefulness of oligonucleotidecompositions in various applications and indications, those skilled inthe art have endeavoured to develop modifications of oligonucleotidestructures that may have preferred or desirable characteristics orattributes as compared with naturally-occurring oligonucleotidemolecules, for example as used in particular applications andindications. Exemplary such modifications are described below.

WO2010/141471 (herein “Traversa I”) teaches the modification ofdifferent types of nucleic acid constructs modified to have a reducednet polyanionic charge. WO2010/039543 (herein “Travera II”) teachescompositions and methods for making neutral polynucleotides (NNs) withreduced polyanionic charge. WO2008/008476 (herein, “Traversa III”)describes the synthesis of SATE (Imbach-type) phosphate prodrugs.Traversa I, II, and III do not teach chirally controlledoligonucleotides, compositions thereof, and methods of making and usingthe same, as described by the present invention.

WO2010/072831 (herein “Girindus et al.”) also teaches the modificationof oligonucleotides. In particular, Girindus et al. teaches the use ofsulfurization reagents to generate phosphorothioate triesters asprodrugs. Girindus et al. does not teach chirally controlledoligonucleotides, compositions thereof, and methods of making and usingthe same, as described by the present invention.

Similarly, WO2004/085454 (herein “Avecia I”) teaches the preparation ofphosphorothioate oligonucleotides through, e.g., transient silylation ofpoly-H-phosphonate diesters. WO2001/027126 (herein “Avecia II”) teachesprocesses for the solid phase synthesis of phosphotriesteroligonucleotides by coupling H-phosphonate monomers to a solid supported5′-hydroxyl oligonucleotide and further sulfurization of the resultingH-phosphonte diester into a phosphorothioate triester. The disclosure ofWO2001/064702 (herein “Avecia III”) is similar to Avecia II and furtherdescribes solid-phase synthesis on different solid supports. Avecia I,II, and III do not teach chirally controlled oligonucleotides,compositions thereof, and methods of making and using the same, asdescribed by the present invention.

WO1997/006183 (herein “Chiron”) teaches oligonucleotides with cationicinternucleotide linkages comprising asymmetric phosphorus, such asstereopure amidates. Chiron teaches stereopure oligonucleotides obtainedvia crystallization of a mixture of diastereomers or via resolutionusing, e.g., column chromatography. Chiron does not teach chirallycontrolled oligonucleotides, compositions thereof, and methods of makingand using the same, as described by the present invention.

WO2009/146123 (herein “Spring Bank I”) teaches compositions and methodsfor treating viral infections using substituted phosphateoligonucleotides and phosphorothioate triesters. WO2007/070598 (herein“Spring Bank II”) teaches phosphotriester prodrugs as antiviral nucleicacids and teaches the synthesis of phosphorothioate prodrugs. SpringBank I and II do not teach chirally controlled oligonucleotides,compositions thereof, and methods of making and using the same, asdescribed by the present invention.

EP0779893 (herein “Hybridon”) teaches lipophilic prodrugs for theincreased cellular uptake of antisense oligonucleotides and observesthat Rp and Sp phosphorothioates and phosphorothioate triester dimerscan have different enzymatic stability properties. Hybridon does notteach chirally controlled oligonucleotides, compositions thereof, andmethods of making and using the same, as described by the presentinvention.

WO1997/047637 (herein “Imbach I”) teaches generally the Imbach “SATE”(S-acyl thioethyl) prodrug oligonucleotide compositions and methods.Imbach I describes, for example, bioreversible phosphotriester prodrugsand the preparation of certain prodrug oligonucleotides usingpost-synthestic alkylation or prodrug-group-containing phosphoramidites.U.S. Pat. No. 6,124,445 (herein “Imbach II”) teaches modified antisenseand chimeric prodrug oligonucleotides. Imbach I and II do not teachchirally controlled oligonucleotides, compositions thereof, and methodsof making and using the same, as described by the present invention.

WO2006/065751 (herein “Beaucage”) teaches CpG oligonucleotidephosphorothioate prodrugs that comprise thermolabile substituents (whichsubstituents are introduced via a phosphoramidite monomer), andapplications thereof. Beaucage does not teach chirally controlledoligonucleotides, compositions thereof, and methods of making and usingthe same, as described by the present invention.

Takeshi Wada et al. developed novel methods for the stereo-controlledsynthesis of P-chiral nucleic acids using amidite chiral auxiliaries(JP4348077, WO2005/014609, WO2005/092909, and WO2010/064146,cumulatively referred to herein as “Wada I”). In particular,WO2010/064146 (referred to herein as “Wada II”) discloses methods forsynthesizing phosphorus atom-modified nucleic acids wherein thestereochemical configuration at phosphorus is controlled. However, themethods of Wada II are limited in that they do not provide forindividual P-modification of each chiral linkage phosphorus in acontrolled and designed manner. That is, the methods for P-modifiedlinkages of Wada II provide for the generation of a condensedintermediate poly H-phosphonate oligonucleotide strand that, once builtto a desired length, is mass modified at the linkage phosphorus toprovide, e.g., a desired phosphorothioate diester, phosphoramidate orboranophosphate or other such phosphorus atom-modified nucleic acids(referred to as Route B in the document—Scheme 6, page 36). Furthermore,the H-phosphonate oligonucleotide strands of Wada II are of shorterlengths (e.g., dimer trimer, or tetramer). Combined with the fact thatthere is no capping step in route B, which generally presents low crudepurity as a result of the accumulation of “n-1”-type byproducts, theWada II route contains limitations in regards of the synthesis of longeroligonucleotides. While Wada II contemplates generally that a particularoligonucleotide could be envisaged to contain different modifications ateach linkage phosphorus, Wada II does not describe or suggest methodsfor controlled iterative installation of such modifications, as aredescribed herein. To the extent that Wada II depicts a synthetic cyclethat does not require an H-phosphonate intermediate oligonucleotide tobe completely assembled prior to modification at the linkage phosphorus(therein referred to as Route A, page 35, Scheme 5, “Synthesis of anucleic acid comprising a chiral X-phosphonate moiety of Formula 1 viaRoute A”), this general disclosure does not teach certain key steps thatare required to install certain P-modifications, as provided by thepresent invention, and especially not with any degree of efficiency andversatility such that this cycle would be useful in the synthesis ofchirally controlled P-modified oligonucleotides, and especiallyoligonucleotides of longer lengths.

At least one such inefficiency of Wada II is noted by Wada et al. inWO2012/039448 (herein “Wada III”). Wada III teaches novel chiralauxiliaries for use in Wada II methods to produce H-phosphonateoligonucleotides that, once built, can be subsequently modified toprovide, inter alia, phosphorothioates and the like. Wada et al. observein Wada III that the four types of chiral auxiliaries disclosed in WadaII formed strong bonds with phosphorus at the linkage phosphorus andthus did not allow for efficient removal. Wada III notes that removal ofthe Wada II chiral auxiliaries required harsh conditions, whichconditions were prone to compromising the integrity of the productoligonucleotide. Wada III observes that this is especially problematicwhen synthesizing long chain oligonucleotides for at least the reasonthat as the degradation reaction(s) proceed, additional byproducts aregenerated that can further react with and degrade the productoligonucleotide. Wada III therefore provides chiral auxiliaries that canbe more efficiently cleaved from the oligonucleotide under mild acidicconditions by way of an S_(N)1 mechanism releasing the H-phosphonateinternucleotide linkage (route B), or under relatively mild basicconditions, by a β-elimation pathway.

One of skill in the chemical and synthetic arts will immediatelyappreciate the complexities associated with generating chirallycontrolled oligonucleotides such as those provided by the presentinvention. For instance, in order to synthesize and isolate a chirallycontrolled oligonucleotide, conditions for each monomer addition must bedesigned such that (1) the chemistry is compatible with every portion ofthe growing oligonucleotide; (2) the byproducts generated during eachmonomer addition do not compromise the structural and stereochemicalintegrity of the growing oligonucleotide; and (3) the crude finalproduct composition is a composition which allows for isolation of thedesired chirally controlled oligonucleotide product.

Oligonucleotide phosphorothioates have shown therapeutic potential(Stein et al., Science (1993), 261:1004-12; Agrawal et al., AntisenceRes. and Dev. (1992), 2:261-66; Bayever et al., Antisense Res. and Dev.(1993), 3:383-390). Oligonucleotide phosphorothioates prepared withoutregard to the stereochemistry of the phosphorothioate exist as a mixtureof 2 ^(n) diastereomers, where n is the number of internucleotidephosphorothioates linkages. The chemical and biological properties ofthese diastereomeric phosphorothioates can be distinct. For example,Wada et al (Nucleic Acids Symposium Series No. 51 p. 119-120;doi:10.1093/nass/nrm060) found that stereodefined-(Rp)-(Ups)9U/(Ap)9Aduplex showed a higher Tm value than that of natural-(Up)9U/(Ap)9A andstereodefined-(Sp)-(Ups)9U did not form a duplex. In another example, ina study by Tang et al., (Nucleosides Nucleotides (1995), 14:985-990)stereopure Rp-oligodeoxyribonucleoside phosphorothioates were found topossess lower stability to nucleases endogenous to human serum that theparent oligodeoxyribonucleoside phosphorothioates with undefinedphosphorus chirality.

Chirally Controlled Oligonucleotides and Chirally ControlledOligonucleotide Compositions

The present invention provides chirally controlled oligonucleotides, andchirally controlled oligonucleotide compositions which are of high crudepurity and of high diastereomeric purity. In some embodiments, thepresent invention provides chirally controlled oligonucleotides, andchirally controlled oligonucleotide compositions which are of high crudepurity. In some embodiments, the present invention provides chirallycontrolled oligonucleotides, and chirally controlled oligonucleotidecompositions which are of high diastereomeric purity.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition comprising oligonucleotidesdefined by having:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers.

In some embodiments, the present invention provides chirally controlledoligonucleotide composition of oligonucleotides in that the compositionis enriched, relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide type.In some embodiments, the present invention provides chirally controlledoligonucleotide composition of oligonucleotides in that the compositionis enriched, relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide typethat share:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type.

In some embodiments, as understood by a person having ordinary skill inthe art, in a substantially racemic (or chirally uncontrolled)preparation of oligonucleotides, all or most coupling steps are notchirally controlled in that the coupling steps are not specificallyconducted to provide enhanced stereoselectivity. An exemplarysubstantially racemic preparation of oligonucleotides is the preparationof phosphorothioate oligonucleotides through sulfurizing phosphitetriesters with either tteraethylthiuram disulfide or (TETD) or 3H-1,2-bensodithiol-3-one 1, 1-dioxide (BDTD), a well-known process in theart. In some embodiments, substantially racemic preparation ofoligonucleotides provides substantially racemic oligonucleotidecompositions (or chirally uncontrolled oligonucleotide compositions).

In some embodiments, a chirally controlled oligonucleotide compositionis a substantially pure preparation of a oligonucleotide type in thatoligonucleotides in the composition that are not of the oligonucleotidetype are impurities form the preparation process of said oligonucleotidetype, in some case, after certain purification procedures.

In some embodiments, the present invention provides oligonucleotidescomprising one or more diastereomerically pure internucleotidic linkageswith respect to the chiral linkage phosphorus. In some embodiments, thepresent invention provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I. In some embodiments, the present invention providesoligonucleotides comprising one or more diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus,and one or more phosphate diester linkages. In some embodiments, thepresent invention provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I, and one or more phosphate diester linkages. In someembodiments, the present invention provides oligonucleotides comprisingone or more diastereomerically pure internucleotidic linkages having thestructure of formula I-c, and one or more phosphate diester linkages. Insome embodiments, such oligonucleotides are prepared by usingstereoselective oligonucleotide synthesis, as described in thisapplication, to form pre-designed diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus.For instance, in one exemplary oligonucleotide of (Rp/Sp, Rp/Sp, Rp/Sp,Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGs1Cs1As1CsC] (SEQ ID NO: 63), thefirst three internucleotidic linkages are constructed using traditionaloligonucleotide synthesis method, and the diastereomerically pureinternucleotidic linkages are constructed with stereochemical control asdescribed in this application. Exemplary internucleotidic linkages,including those having structures of formula I, are further describedbelow.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In certain embodiments, the present invention provides achirally controlled oligonucleotide, wherein at least two individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another. In certain embodiments, thepresent invention provides a chirally controlled oligonucleotide,wherein at least two of the individual internucleotidic linkages withinthe oligonucleotide have different P-modifications relative to oneanother, and wherein the chirally controlled oligonucleotide comprisesat least one phosphate diester internucleotidic linkage. In certainembodiments, the present invention provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate diesterinternucleotidic linkage. In certain embodiments, the present inventionprovides a chirally controlled oligonucleotide, wherein at least two ofthe individual internucleotidic linkages within the oligonucleotide havedifferent P-modifications relative to one another, and wherein thechirally controlled oligonucleotide comprises at least onephosphorothioate triester internucleotidic linkage. In certainembodiments, the present invention provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterinternucleotidic linkage.

In certain embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages independently having the structure of formulaI:

wherein each variable is as defined and described below. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising one or more modified internucleotidiclinkages of formula I, and wherein individual internucleotidic linkagesof formula I within the oligonucleotide have different P-modificationsrelative to one another. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising one or moremodified internucleotidic linkages of formula I, and wherein individualinternucleotidic linkages of formula I within the oligonucleotide havedifferent —X—L—R¹ relative to one another. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomprising one or more modified internucleotidic linkages of formula I,and wherein individual internucleotidic linkages of formula I within theoligonucleotide have different X relative to one another. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising one or more modified internucleotidiclinkages of formula I, and wherein individual internucleotidic linkagesof formula I within the oligonucleotide have different —L—R¹ relative toone another.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry relative to one another, and wherein at least a portionof the structure of the chirally controlled oligonucleotide ischaracterized by a repeating pattern of alternating stereochemisty.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another, in that they have different Xatoms in their —XLR¹ moieties, and/or in that they have different Lgroups in their —XLR¹ moieties, and/or that they have different R¹ atomsin their —XLR¹ moieties.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to one anotherand the oligonucleotide has a structure represented by the followingformula:[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny]wherein:each R^(B) independently represents a block of nucleotide units havingthe R configuration at the linkage phosphorus;each S^(B) independently represents a block of nucleotide units havingthe S configuration at the linkage phosphorus;each of n1-ny is zero or an integer, with the requirement that at leastone odd n and at least one even n must be non-zero so that theoligonucleotide includes at least two individual internucleotidiclinkages with different stereochemistry relative to one another; andwherein the sum of n1-ny is between 2 and 200, and in some embodimentsis between a lower limit selected from the group consisting of 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more and an upper limit selected from the group consisting of5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upperlimit being larger than the lower limit.

In some such embodiments, each n has the same value; in someembodiments, each even n has the same value as each other even n; insome embodiments, each odd n has the same value each other odd n; insome embodiments, at least two even ns have different values from oneanother; in some embodiments, at least two odd ns have different valuesfrom one another.

In some embodiments, at least two adjacent ns are equal to one another,so that a provided oligonucleotide includes adjacent blocks of Sstereochemistry linkages and R stereochemistry linkages of equallengths. In some embodiments, provided oligonucleotides includerepeating blocks of S and R stereochemistry linkages of equal lengths.In some embodiments, provided oligonucleotides include repeating blocksof S and R stereochemistry linkages, where at least two such blocks areof different lengths from one another; in some such embodiments each Sstereochemistry block is of the same length, and is of a differentlength from each R stereochemistry length, which may optionally be ofthe same length as one another.

In some embodiments, at least two skip-adjacent ns are equal to oneanother, so that a provided oligonucleotide includes at least two blocksof linkages of a first steroechemistry that are equal in length to oneanother and are separated by a block of linkages of the otherstereochemistry, which separating block may be of the same length or adifferent length from the blocks of first steroechemistry.

In some embodiments, ns associated with linkage blocks at the ends of aprovided oligonucleotide are of the same length. In some embodiments,provided oligonucleotides have terminal blocks of the same linkagestereochemistry. In some such embodiments, the terminal blocks areseparated from one another by a middle block of the other linkagestereochemistry.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny] is a stereoblockmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny] is a stereoskipmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4...S^(B)nxR^(B)ny] is a stereoaltmer. Insome embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny] is a gapmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny] is of any of theabove described patterns and further comprises patterns ofP-modifications. For instance, in some embodiments, a providedoligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . .S^(B)nxR^(B)ny] and is a stereoskipmer and P-modification skipmer. Insome embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny] and is astereoblockmer and P-modification altmer. In some embodiments, aprovided oligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . .S^(B)nxR^(B)ny] and is a stereoaltmer and P-modification blockmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4. . . S^(B)nxR^(B)ny] is a chirallycontrolled oligonucleotide comprising one or more modifiedinternuceotidic linkages independently having the structure of formulaI:

wherein:P* is an asymmetric phosphorus atom and is either Rp or Sp;W is O, S or Se;each of X, Y and Z is independently —O—, —S—, —N(—L—R¹), or L;L is a covalent bond or an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—;each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:

two R′ on the same nitrogen are taken together with their interveningatoms to form an optionally substituted heterocyclic or heteroaryl ring,or

two R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring;

—Cy— is an optionally substituted bivalent ring selected from phenylene,carbocyclylene, arylene, heteroarylene, or heterocyclylene;

each R is independently hydrogen, or an optionally substituted groupselected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, orheterocyclyl; and each —

— independently represents a connection to a nucleoside.

In some embodiments, a chirally controlled oligonucleotide comprises oneor more modified internucleotidic phosphorus linkages. In someembodiments, a chirally controlled oligonucleotide comprises, e.g., aphosphorothioate or a phosphorothioate triester linkage. In someembodiments, a chirally controlled oligonucleotide comprises aphosphorothioate triester linkage. In some embodiments, a chirallycontrolled oligonucleotide comprises at least two phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least three phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least four phosphorothioate triester linkages. In someembodiments, a chirally controlled oligonucleotide comprises at leastfive phosphorothioate triester linkages. Exemplary such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a chirally controlled oligonucleotide comprisesdifferent internucleotidic phosphorus linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least one modifiedinternucleotidic linkage. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast two phosphorothioate triester linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast five phosphorothioate triester linkages. Exemplary such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a phosphorothioate triester linkage comprises achiral auxiliary, which, for example, is used to control thestereoselectivity of a reaction. In some embodiments, a phosphorothioatetriester linkage does not comprise a chiral auxiliary. In someembodiments, a phosphorothioate triester linkage is intentionallymaintained until and/or during the administration to a subject.

In some embodiments, a chirally controlled oligonucleotide is linked toa solid support. In some embodiments, a chirally controlledoligonucleotide is cleaved from a solid support.

In some embodiments, a chirally controlled oligonucleotide comprises atleast one phosphate diester internucleotidic linkage and at least twoconsecutive modified internucleotidic linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least two consecutivephosphorothioate triester internucleotidic linkages.

In some embodiments, a chirally controlled oligonucleotide is ablockmer. In some embodiments, a chirally controlled oligonucleotide isa stereoblockmer. In some embodiments, a chirally controlledoligonucleotide is a P-modification blockmer. In some embodiments, achirally controlled oligonucleotide is a linkage blockmer.

In some embodiments, a chirally controlled oligonucleotide is an altmer.In some embodiments, a chirally controlled oligonucleotide is astereoaltmer. In some embodiments, a chirally controlled oligonucleotideis a P-modification altmer. In some embodiments, a chirally controlledoligonucleotide is a linkage altmer.

In some embodiments, a chirally controlled oligonucleotide is a unimer.In some embodiments, a chirally controlled oligonucleotide is astereounimer. In some embodiments, a chirally controlled oligonucleotideis a P-modification unimer. In some embodiments, a chirally controlledoligonucleotide is a linkage unimer.

In some embodiments, a chirally controlled oligonucleotide is a gapmer.

In some embodiments, a chirally controlled oligonucleotide is a skipmer.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages independently having the structure of formulaI:

wherein:P* is an asymmetric phosphorus atom and is either Rp or Sp;W is O, S or Se;each of X, Y and Z is independently —O—, —S—, —N(—L—R¹), or L;L is a covalent bond or an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—;each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:

two R′ on the same nitrogen are taken together with their interveningatoms to form an optionally substituted heterocyclic or heteroaryl ring,or

two R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring;

—Cy— is an optionally substituted bivalent ring selected from phenylene,carbocyclylene, arylene, heteroarylene, or heterocyclylene;

each R is independently hydrogen, or an optionally substituted groupselected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, orheterocyclyl; and

each —

— independently represents a connection to a nucleoside.

As defined generally above and herein, P* is an asymmetric phosphorusatom and is either Rp or Sp. In some embodiments, P* is Rp. In otherembodiments, P* is Sp. In some embodiments, an oligonucleotide comprisesone or more internucleotidic linkages of formula I wherein each P* isindependently Rp or Sp. In some embodiments, an oligonucleotidecomprises one or more internucleotidic linkages of formula I whereineach P* is Rp. In some embodiments, an oligonucleotide comprises one ormore internucleotidic linkages of formula I wherein each P* is Sp. Insome embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein P* is Rp. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein P* is Sp. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein P* is Rp, and at least one internucleotidic linkage offormula I wherein P* is Sp.

As defined generally above and herein, W is O, S, or Se. In someembodiments, W is O. In some embodiments, W is S. In some embodiments, Wis Se. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein W is O. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein W is S. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein W is Se.

As defined generally above and herein, each R is independently hydrogen,or an optionally substituted group selected from C₁-C₆ aliphatic,phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is hydrogen. In some embodiments, R is anoptionally substituted group selected from C₁-C₆ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R is an optionally substituted C₁-C₆ alkyl. In someembodiments, R is optionally substituted, linear or branched hexyl. Insome embodiments, R is optionally substituted, linear or branchedpentyl. In some embodiments, R is optionally substituted, linear orbranched butyl. In some embodiments, R is optionally substituted, linearor branched propyl. In some embodiments, R is optionally substitutedethyl. In some embodiments, R is optionally substituted methyl.

In some embodiments, R is optionally substituted phenyl. In someembodiments, R is substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R is optionally substituted carbocyclyl. In someembodiments, R is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R is optionally substituted monocyclic carbocyclyl. In someembodiments, R is optionally substituted cycloheptyl. In someembodiments, R is optionally substituted cyclohexyl. In someembodiments, R is optionally substituted cyclopentyl. In someembodiments, R is optionally substituted cyclobutyl. In someembodiments, R is an optionally substituted cyclopropyl. In someembodiments, R is optionally substituted bicyclic carbocyclyl.

In some embodiments, R is an optionally substituted aryl. In someembodiments, R is an optionally substituted bicyclic aryl ring.

In some embodiments, R is an optionally substituted heteroaryl. In someembodiments, R is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R is a substituted 5-6membered monocyclic heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R is an optionallysubstituted 6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Exemplary R groupsinclude optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Exemplary Rgroups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having 1 heteroatom independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted indolyl. In some embodiments, R is an optionally substitutedazabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionallysubstituted 5,6-fused heteroaryl ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted azaindolyl. In some embodiments, R is anoptionally substituted benzimidazolyl. In some embodiments, R is anoptionally substituted benzothiazolyl. In some embodiments, R is anoptionally substituted benzoxazolyl. In some embodiments, R is anoptionally substituted indazolyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is an optionally substituted quinolinyl.In some embodiments, R is an optionally substituted isoquinolinyl.According to one aspect, R is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is a quinazoline ora quinoxaline.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 6 membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atom.

In certain embodiments, R is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, Ris an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is an optionally substituted indolinyl. In someembodiments, R is an optionally substituted isoindolinyl. In someembodiments, R is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

As defined generally above and herein, each R′ is independently—R,—C(O)R, —CO₂R, or —SO₂R, or:

two R′ on the same nitrogen are taken together with their interveningatoms to form an optionally substituted heterocyclic or heteroaryl ring,or

two R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring.

In some embodiments, R′ is —R, —C(O)R, —CO₂R, or —SO₂R, wherein R is asdefined above and described herein.

In some embodiments, R′ is —R, wherein R is as defined and describedabove and herein. In some embodiments, R′ is hydrogen.

In some embodiments, R′ is —C(O)R, wherein R is as defined above anddescribed herein. In some embodiments, R′ is —CO₂R, wherein R is asdefined above and described herein. In some embodiments, R′ is —SO₂R,wherein R is as defined above and described herein.

In some embodiments, two R′ on the same nitrogen are taken together withtheir intervening atoms to form an optionally substituted heterocyclicor heteroaryl ring. In some embodiments, two R′ on the same carbon aretaken together with their intervening atoms to form an optionallysubstituted aryl, carbocyclic, heterocyclic, or heteroaryl ring.

As defined generally above and herein, —Cy— is an optionally substitutedbivalent ring selected from phenylene, carbocyclylene, arylene,heteroarylene, or heterocyclylene.

In some embodiments, —Cy— is optionally substituted phenylene. In someembodiments, —Cy— is optionally substituted carbocyclylene. In someembodiments, —Cy— is optionally substituted arylene. In someembodiments, —Cy— is optionally substituted heteroarylene. In someembodiments, —Cy— is optionally substituted heterocyclylene.

As defined generally above and herein, each of X, Y and Z isindependently —O—, —S—, —N(—L—R¹)—, or L, wherein each of L and R¹ isindependently as defined above and described below.

In some embodiments, X is —O—. In some embodiments, X is —S—. In someembodiments, X is —O— or —S—. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein Xis —O—. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein X is —S—. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein X is —O—, and at least one internucleotidiclinkage of formula I wherein X is —S—. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein X is —O—, and at least one internucleotidic linkage offormula I wherein X is —S—, and at least one internucleotidic linkage offormula I wherein L is an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—.

In some embodiments, X is —N(—L—R¹)—. In some embodiments, X is —N(R¹)—.In some embodiments, X is —N(R′)—. In some embodiments, X is —N(R)—. Insome embodiments, X is —NH—.

In some embodiments, X is L. In some embodiments, X is a covalent bond.In some embodiments, X is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, ═C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, X is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, X is methylene.

In some embodiments, Y is —O—. In some embodiments, Y is —S—.

In some embodiments, Y is —N(—L—R¹)—. In some embodiments, Y is —N(R¹)—.In some embodiments, Y is —N(R′)—. In some embodiments, Y is —N(R)—. Insome embodiments, Y is —NH—.

In some embodiments, Y is L. In some embodiments, Y is a covalent bond.In some embodiments, Y is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Y is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Y is methylene.

In some embodiments, Z is —O—. In some embodiments, Z is —S—.

In some embodiments, Z is —N(—L—R¹)—. In some embodiments, Z is —N(R¹)—.In some embodiments, Z is —N(R′)—. In some embodiments, Z is —N(R)—. Insome embodiments, Z is —NH—.

In some embodiments, Z is L. In some embodiments, Z is a covalent bond.In some embodiments, Z is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Z is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Z is methylene.

As defined generally above and herein, L is a covalent bond or anoptionally substituted, linear or branched C₁-C₁₀ alkylene, wherein oneor more methylene units of L are optionally and independently replacedby an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—.

In some embodiments, L is a covalent bond. In some embodiments, L is anoptionally substituted, linear or branched C₁-C₁₀ alkylene, wherein oneor more methylene units of L are optionally and independently replacedby an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—.

In some embodiments, L has the structure of —L¹—V—, wherein: L¹ is anoptionally substituted group selected from

C₁-C₆ alkylene, C₁-C₆ alkenylene, carbocyclylene, arylene, C₁-C₆heteroalkylene, heterocyclylene, and heteroarylene;V is selected from —O—, —S—, —NR′—, C(R′)₂, —S—S—, —B—S—S—C—,

or an optionally substituted group selected from C₁-C₆ alkylene,arylene, C₁-C₆ heteroalkylene, heterocyclylene, and heteroarylene;A is ═O, ═S, ═NR′, or ═C(R′)₂;each of B and C is independently —O—, —S—, —NR′—, —C(R′)₂—, or anoptionally substituted group selected from C₁-C₆ alkylene,carbocyclylene, arylene, heterocyclylene, or heteroarylene; and each R′is independently as defined above and described herein.

In some embodiments, L¹ is

In some embodiments, L¹ is

wherein Ring Cy′ is an optionally substituted arylene, carbocyclylene,heteroarylene, or heterocyclylene. In some embodiments, L¹ is optionallysubstituted

In some embodiments, L¹ is

In some embodiments, L¹ is connected to X. In some embodiments, L¹ is anoptionally substituted group selected from

and the sulfur atom is connect to V. In some embodiments, L¹ is anoptionally substituted group selected from

and the carbon atom is connect to X.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, carbocyclic,heteroaryl or heterocyclic ring; and each R′ is independently as definedabove and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;

is a single or double bond; andthe two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂—(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR';D is ═N, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;D is ═N, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring; and each R′ isindependently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR';

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring;and each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂),═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃); andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring; and each R′ isindependently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring; and each R′ isindependently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′— or —C(R′)₂—;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—,or ═C(CF₃)—; andR′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein:E is —O—, —S—, —NR′ or —C(R′)₂—;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—, or ═C(CF₃)—; andeach R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,═C(CO₂-(C₁-C₆ aliphatic))—,or ═C(CF₃)—; andR′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein:

is a single or double bond; andthe two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:G is —O—, —S—, or —NR′;

is a single or double bond; andthe two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

As defined generally above and herein, E is —O—, —S—, —NR′— or —C(R′)₂—,wherein each R′ independently as defined above and described herein. Insome embodiments, E is —O—, —S—, or —NR′—. In some embodiments, E is—O—, —S—, or —NH—. In some embodiments, E is —O—. In some embodiments, Eis —S—. In some embodiments, E is —NH—.

As defined generally above and herein, G is —O—, —S—, or —NR′, whereineach R′ independently as defined above and described herein. In someembodiments, G is —O—, —S—, or —NH—. In some embodiments, G is —O—. Insome embodiments, G is —S—. In some embodiments, G is —NH—.

In some embodiments, L is —L³—G, wherein:

L³ is an optionally substituted C₁-C₅ alkylene or alkenylene, whereinone or more methylene units are optionally and independently replaced by—O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)₂—, or

andwherein each of G, R′ and Ring Cy′ is independently as defined above anddescribed herein.

In some embodiments, L is —L³—S—, wherein L³ is as defined above anddescribed herein. In some embodiments, L is —L³—O—, wherein L³ is asdefined above and described herein. In some embodiments, L is—L³—N(R′)—, wherein each of L³ and R′ is independently as defined aboveand described herein. In some embodiments, L is —L³—NH—, wherein each ofL³ and R′ is independently as defined above and described herein.

In some embodiments, L³ is an optionally substituted C₅ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Ring Cy′ is independently as defined above anddescribed herein. In some embodiments, L³ is an optionally substitutedC₅ alkylene. In some embodiments, —L³—G— is

In some embodiments, L³ is an optionally substituted C₄ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, —L³—G— is

In some embodiments, L³ is an optionally substituted C₃ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, —L³—G— is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L³ is an optionally substituted C₂ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, —L³—G— is

wherein each of G and Cy′ is independently as defined above anddescribed herein. In some embodiments, L is

In some embodiments, L is —L⁴—G—, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; and G is as defined above and describedherein. In some embodiments, L is —L⁴—G—, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; G is as defined above and described herein;and G is connected to R¹. In some embodiments, L is —L⁴—G—, wherein L⁴is an optionally substituted methylene; G is as defined above anddescribed herein; and G is connected to R¹. In some embodiments, L is—L⁴—G—, wherein L⁴ is methylene; G is as defined above and describedherein; and G is connected to R¹. In some embodiments, L is —L⁴—G—,wherein L⁴ is an optionally substituted —(CH₂)₂—; G is as defined aboveand described herein; and G is connected to R¹. In some embodiments, Lis —L⁴—G—, wherein L⁴ is —(CH₂)₂—; G is as defined above and describedherein; and G is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein the sulfur atom is connected to R¹. In some embodiments, L is

wherein the oxygen atom is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein.

In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3)is an optionally substituted, linear or branched, C₁-C₉ alkylene,wherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy- , —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each of R′ and -—Cy— isindependently as defined above and described herein. In someembodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is anoptionally substituted C₁-C₆ alkylene. In some embodiments, L is—S—R^(L3)— or —S—C(O)—R^(L3), wherein R^(L3) is an optionallysubstituted C₁C₆ alkenylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkylene wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkenylene,arylene, or heteroarylene. In some embodiments, In some embodiments,R^(L3) is an optionally substituted —S—(C₁-C₆ alkenylene)—, —S—(C₁-C₆alkylene)-, —S—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-,—S—CO—arylene(C₁-C₆ alkylene)-, or —S—CO—(C₁-C₆ alkylene)-arylene-(C₁-C₆alkylene)—.

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments,

In some embodiments, the sulfur atom in the L embodiments describedabove and herein is connected to X. In some embodiments, the sulfur atomin the L embodiments described above and herein is connected to R¹.

As defined generally above and herein, R¹ is halogen, R, or anoptionally substituted C₁-C₅₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—, wherein each variable is independently as defined above anddescribed herein. In some embodiments, R¹ is halogen, R, or anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—, wherein each variable is independently as defined above anddescribed herein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is halogen.In some embodiments, R¹ is —F. In some embodiments, R¹ is —Cl. In someembodiments, R¹ is —Br. In some embodiments, R¹ is —I.

In some embodiments, R¹ is R wherein R is as defined above and describedherein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is anoptionally substituted group selected from C₁-C₅₀ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R¹ is an optionally substituted C₁-C₆ alkyl. In someembodiments, R¹ is optionally substituted, linear or branched hexyl. Insome embodiments, R¹ is optionally substituted, linear or branchedpentyl. In some embodiments, R¹ is optionally substituted, linear orbranched butyl. In some embodiments, R¹ is optionally substituted,linear or branched propyl. In some embodiments, R¹ is optionallysubstituted ethyl. In some embodiments, R¹ is optionally substitutedmethyl.

In some embodiments, R¹ is optionally substituted phenyl. In someembodiments, R¹ is substituted phenyl. In some embodiments, R¹ isphenyl.

In some embodiments, R¹ is optionally substituted carbocyclyl. In someembodiments, R¹ is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R¹ is optionally substituted monocyclic carbocyclyl. Insome embodiments, R¹ is optionally substituted cycloheptyl. In someembodiments, R¹ is optionally substituted cyclohexyl. In someembodiments, R¹ is optionally substituted cyclopentyl. In someembodiments, R¹ is optionally substituted cyclobutyl. In someembodiments, R¹ is an optionally substituted cyclopropyl. In someembodiments, R¹ is optionally substituted bicyclic carbocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ polycyclichydrocarbon. In some embodiments, R¹ is an optionally substituted C₁-C₅₀polycyclic hydrocarbon wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein eachvariable is independently as defined above and described herein. In someembodiments, R¹ is optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is optionally substituted

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphaticcomprising one or more optionally substituted polycyclic hydrocarbonmoieties. In some embodiments, R¹ is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted polycyclichydrocarbon moieties, wherein one or more methylene units are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic comprisingone or more optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted aryl. In someembodiments, R¹ is an optionally substituted bicyclic aryl ring.

In some embodiments, R¹ is an optionally substituted heteroaryl. In someembodiments, R¹ is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R¹ is a substituted5-6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an unsubstituted 5-6 membered monocyclic heteroarylring having 1-3 heteroatoms independently selected from nitrogen,sulfur, or oxygen.

In some embodiments, R¹ is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R¹ is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R¹ is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R¹ is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Exemplary R¹groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl .

In some embodiments, R¹ is a is a 6-membered heteroaryl ring having1-3nitrogen atoms. In other embodiments, R¹ is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R¹ is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R¹ is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Exemplary R¹groups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R¹ is an optionally substituted5,6-fused heteroaryl ring having 1 heteroatom independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted indolyl. In some embodiments, R¹ is an optionallysubstituted azabicyclo[3.2.1]octanyl. In certain embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted azaindolyl. In someembodiments, R¹ is an optionally substituted benzimidazolyl. In someembodiments, R¹ is an optionally substituted benzothiazolyl. In someembodiments, R¹ is an optionally substituted benzoxazolyl. In someembodiments, R¹ is an optionally substituted indazolyl. In certainembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 3 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In certain embodiments, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R¹ is an optionally substituted quinolinyl.In some embodiments, R¹ is an optionally substituted isoquinolinyl.According to one aspect, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is a quinazoline ora quinoxaline.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 6 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atoms.

In certain embodiments, R¹ is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹ isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R¹is an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹ is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R¹ is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R¹ is an optionally substituted indolinyl. In someembodiments, R¹ is an optionally substituted isoindolinyl. In someembodiments, R¹ is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R¹ is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently asdefined above and described herein. In some embodiments, R¹ is anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionally —Cy—,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —OC(O)—, or —C(O)O—, wherein eachR′ is independently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —OC(O)—, or—C(O)O—, wherein each R′ is independently as defined above and describedherein.

In some embodiments, R¹ is

In some embodiments, R¹ is CH₃—,

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to L. Exemplary such R¹ groups aredepicted below:

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to L. Ecemplary such R¹ _(g)roups aredepicted below:

In some embodiments, R¹ is —S—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′—and—Cy— is independently as defined above and described herein. In someembodiments, R¹ is —S—R^(L2), wherein the sulfur atom is connected withthe sulfur atom in L group.

In some embodiments, R¹ is —C(O)—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substitutedC_(l)-C₆ alkylene, C_(l)-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—, —O—, —S—,—S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and—Cy— is independently as defined above and described herein. In someembodiments, R¹ is —C(O)R^(L2), wherein the carbonyl group is connectedwith G in L group. In some embodiments, R¹ is —C(O)R^(L2), wherein thecarbonyl group is connected with the sulfur atom in L group.

In some embodiments, R^(L2) is optionally substituted C₁-C₉ aliphatic.In some embodiments, R^(L2) is optionally substituted C₁-C₉ alkyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkenyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkynyl. Insome embodiments, R^(L2) is an optionally substituted C₁-C₉ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by —Cy— or —C(O)—. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by —Cy—. In some embodiments,R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted heterocycylene. In some embodiments, R^(L2) is anoptionally substituted C₁-C₉ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted arylene. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substitutedheteroarylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₃-C₁₀carbocyclylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein two methylene units are optionally andindependently replaced by —Cy— or —C(O)—. In some embodiments, R^(L2) isan optionally substituted C₁-C₉ aliphatic wherein two methylene unitsare optionally and independently replaced by —Cy— or —C(O)—. ExemplaryR^(L2) groups are depicted below:

In some embodiments, R¹ is hydrogen, or an optionally substituted groupselected from

—S—(C₁-C₁₀ aliphatic), C₁C₁₀ aliphatic, aryl, C₁-C₆ heteroalkyl,heteroaryl and heterocyclyl. In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic). In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted group selected from—S— (C₁-C₆ aliphatic), C₁-C₁₀ aliphatic, C₁-C₆ heteroaliphatic, aryl,heterocyclyl and heteroaryl.

In some embodiments, R¹ is

In some embodiments, the sulfur atom in the R¹ embodiments describedabove and herein is connected with the sulfur atom, G, E, or —C(O)—moiety in the L embodiments described above and herein. In someembodiments, the —C(O)— moiety in the R¹ embodiments described above andherein is connected with the sulfur atom, G, E, or —C(O)— moiety in theL embodiments described above and herein.

In some embodiments, —L—R¹ is any combination of the L embodiments andR¹ embodiments described above and herein.

In some embodiments, —L—R¹ is —L³—G—R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, —L—R¹ is L⁴—G—R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, —L—R¹ is L³—G—S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, —L—R¹ is L³—G—C(O)—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, —L—R—R¹ is

wherein R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—, and each G is independently as defined above and describedherein.

In some embodiments, —L—R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, —L—R¹ is R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —L—R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, L has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —X—L—R¹ has the structure of:

wherein:the phenyl ring is optionally substituted, andeach of R¹ and X is independently as defined above and described herein.

In some embodiments, —L—R¹ is

In some embodiments, —L—R¹ is:

In some embodiments, —L—R¹ is CH₃—,

In some embodiments, —L—R¹ is

In some embodiments, —L—R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to X. In some embodiments, —L—R¹comprises a terminal —(CH₂)₂— moiety which is connected to X. Exemplarysuch —L—R¹ moieties are depicted below:

In some embodiments, —L—R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to X. In some embodiments, —L—R¹comprises a terminal —(CH₂)— moiety which is connected to X. Exemplarysuch —L—R¹ moieties are depicted below:

In some embodiments, —L—R¹ is

In some embodiments, —L—R¹ is CH₃—,

and X is —S—.

In some embodiments, —L—R¹ is CH₃—,

X is —S—, W is O, Y is —O—, and Z is —O—.

In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, R¹ is

In some embodiments, X is —O—or —S—, and R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, X is —O— or —S—, and R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, L is a covalent bond and —L—R¹ is R¹.

In some embodiments, —L—R¹ is not hydrogen.

In some embodiments, —X—L—R¹ is R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, —X—L—R¹ has the structure of

wherein the

moiety is optionally substituted. In some embodiments, is —X—L—R¹ is

In some embodiments, —X—L—R¹ is

In some embodiments, —X—L—R¹ is

In some embodiments, —X—L—R¹ has the structure of

wherein X′ is O or S, Y′ is —O—, —S—or —NR′—, and the

moiety is optionally substituted. In some embodiments, Y′ is —O—, —S— or—NH—. In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments, —X—L—R¹ has the structure of

wherein X′ is O or S, and the

moiety is optionally substituted. In some embodiments,

In some embodiments, —X—L—R¹ is

wherein the

is optionally substituted. In some embodiments, —X—L—R¹ is

wherein the

is substituted. In some embodiments, —X—L—R¹ is

wherein the

is unsubstituted.

In some embodiments, —X—L—R¹ is R¹—C(O)—S—L^(x)—S—, wherein L^(x) is anoptionally substituted group selected from

In some embodiments, L^(x) is

In some embodiments, —X—L—R¹ is (CH₃)₃C—S—S—L^(x)—S—. In someembodiments, —X—L—R¹ is R¹ —C(═X′)—Y′—C(R)₂—S—L^(x)—S—. In someembodiments, -X-L—R¹ is R—C(═X′)—Y′—CH₂—S—L^(x)—S—. In some embodiments,—X—L—R¹ is

As will be appreciated by a person skilled in the art, many of thegroups described herein are cleavable and can be converted to —X⁻ afteradministration to a subject. In some embodiments, —X—L—R¹ is cleavable.In some embodiments, —X—L—R¹ is —S—L—R¹, and is converted to —S⁻ afteradministration to a subject. In some embodiments, the conversion ispromoted by an enzyme of a subject. As appreciated by a person skilledin the art, methods of determining whether the —S—L—R¹ group isconverted to after administration is widely known and practiced in theart, including those used for studying drug metabolism andpharmacokinetics.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-a:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-b:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I is anphosphorothioate triester linkage having the structure of formula I-c:

wherein:P* is an asymmetric phosphorus atom and is either Rp or Sp;L is a covalent bond or an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂ —, —Cy—, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂, —S(O)₂N(R′)—, N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂ —, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, N(R′)C(O)O—, —OC(O)N(R′)—,S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—;each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:

two R′ on the same nitrogen are taken together with their interveningatoms to form an optionally substituted heterocyclic or heteroaryl ring,or

two R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring;

—Cy— is an optionally substituted bivalent ring selected from phenylene,carbocyclylene, arylene, heteroarylene, or heterocyclylene;

each R is independently hydrogen, or an optionally substituted groupselected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, orheterocyclyl;

each

independently represents a connection to a nucleoside; andR¹ is not —H when L is a covalent bond.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage having the structureof formula I-c is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising one or more phosphate diesterlinkages, and one or more modified internucleotide linkages having theformula of I-a, I-b, or I-c.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage having the structure of formula I-c. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least two phosphorothioate triester linkages having the structure offormula I-c. In some embodiments, the present invention provides achirally controlled oligonucleotide comprising at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages having the structure of formula I-c. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages having the structure of formula I-c. In some embodiments, thepresent invention provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least five phosphorothioate triester linkages having the structure offormula I-c.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9). In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising asequence found in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the saidsequence has over 50% identity with GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9).In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the said sequence has over60% identity with GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9). In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 9), wherein the said sequence has over 70% identity withGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9). In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising asequence found in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the saidsequence has over 80% identity with GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9).In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the said sequence has over90% identity with GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9). In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 9), wherein the said sequence has over 95% identity withGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9). In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9). In some embodiments,the present invention provides a chirally controlled oligonucleotidehaving the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9).

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 9), wherein at least one internucleotidic linkage has thestructure of formula I. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising a sequencefound in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein eachinternucleotidic linkage has the structure of formula I. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 9), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide comprising a sequencefound in GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein eachinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising a sequence found in GCCTCAGTCTGCTTCGCACC (SEQID NO: 9), wherein at least one internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising a sequence found inGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 9), wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present invention provides achirally controlled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each internucleotidiclinkage has the structure of formula I. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide comprising thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present invention provides a chirally controlledoligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 9), wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present invention provides achirally controlled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide comprising the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each internucleotidiclinkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein at least one internucleotidic linkage has achiral linkage phosphorus. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least oneinternucleotidic linkage has the structure of formula I. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein each internucleotidic linkage has the structure of formulaI. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each internucleotidiclinkage has the structure of formula I-c. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at leastone internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein each internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein at least one internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein each internucleotidic linkage is

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein at least one linkage phosphorus is Rp. It isunderstood by a person of ordinary skill in the art that in certainembodiments wherein the chirally controlled oligonucleotide comprises anRNA sequence, each T is independently and optionally replaced with U. Insome embodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein each linkage phosphorus is Rp. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at leastone linkage phosphorus is Sp. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein each linkage phosphorus isSp. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein the oligonucleotide is a blockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein the oligonucleotide is a stereoblockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein the oligonucleotide is a P-modification blockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein the oligonucleotide is a linkage blockmer. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein the oligonucleotide is an altmer. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein theoligonucleotide is a stereoaltmer. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein theoligonucleotide is a P- modification altmer. In some embodiments, thepresent invention provides a chirally controlled oligonucleotide havingthe sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein theoligonucleotide is a linkage altmer. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having thesequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein theoligonucleotide is a unimer. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the oligonucleotide is astereounimer. In some embodiments, the present invention provides achirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the oligonucleotide is aP-modification unimer. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the oligonucleotide is alinkage unimer. In some embodiments, the present invention provides achirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein the oligonucleotide is agapmer. In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein the oligonucleotide is a skipmer.

In some embodiments, the present invention provides a chirallycontrolled oligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC(SEQ ID NO: 9), wherein each cytosine is optionally and independentlyreplaced by 5-methylcytosine. In some embodiments, the present inventionprovides a chirally controlled oligonucleotide having the sequence ofGCCTCAGTCTGCTTCGCACC (SEQ ID NO: 9), wherein at least one cytosine isoptionally and independently replaced by 5-methylcytosine. In someembodiments, the present invention provides a chirally controlledoligonucleotide having the sequence of GCCTCAGTCTGCTTCGCACC (SEQ ID NO:9), wherein each cytosine is optionally and independently replaced by5-methylcytosine.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that one or more nucleotides comprise a phosphorus modificationprone to “autorelease” under certain conditions. That is, under certainconditions, a particular phosphorus modification is designed such thatit self-cleaves from the oligonucleotide to provide, e.g., a phosphatediester such as those found in naturally occurring DNA and RNA. In someembodiments, such a phosphorus modification has a structure of —O—L—R¹,wherein each of L and R¹ is independently as defined above and describedherein. In some embodiments, an autorelease group comprises a morpholinogroup. In some embodiments, an autorelease group is characterized by theability to deliver an agent to the internucleotidic phosphorus linker,which agent facilitates further modification of the phosphorus atom suchas, e.g., desulfurization. In some embodiments, the agent is water andthe further modification is hydrolysis to form a phosphate diester as isfound in naturally occurring DNA and RNA.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that the resulting pharmaceutical properties are improved throughone or more particular modifications at phosphorus. It is welldocumented in the art that certain oligonucleotides are rapidly degradedby nucleases and exhibit poor cellular uptake through the cytoplasmiccell membrane (Poijarvi-Virta et al., Curr. Med. Chem. (2006),13(28);3441-65; Wagner et al., Med. Res. Rev. (2000), 20(6):417-51;Peyrottes et al., Mini Rev. Med. Chem. (2004), 4(4):395-408; Gosselin etal., (1996), 43(1):196-208; Bologna et al., (2002), Antisense & NucleicAcid Drug Development 12:33-41). For instance, Vives et al., (NucleicAcids Research (1999), 27(20):4071-76) found that tert-butyl SATEpro-oligonucleotides displayed markedly increased cellular penetrationcompared to the parent oligonucleotide.

In some embodiments, a modification at a linkage phosphorus ischaracterized by its ability to be transformed to a phosphate diester,such as those present in naturally occurring DNA and RNA, by one or moreesterases, nucleases, and/or cytochrome P450 enzymes, including but notlimited to, those listed in Table 1, below.

TABLE 1 Exemplary enzymes. Family Gene CYP1 CYP1A1, CYP1A2, CYP1B1 CYP2CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19,CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 CYP3CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP4 CYP4A11, CYP4A22, CYP4B1, CYP4F2,CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1 CYP5CYP5A1 CYP7 CYP7A1, CYP7B1 CYP8 CYP8A1 (prostacyclin synthase), CYP8B1(bile acid biosynthesis) CYP11 CYP11A1, CYP11B1, CYP11B2 CYP17 CYP17A1CYP19 CYP19A1 CYP20 CYP20A1 CYP21 CYP21A2 CYP24 CYP24A1 CYP26 CYP26A1,CYP26B1, CYP26C1 CYP27 CYP27A1 (bile acid biosynthesis), CYP27B1(vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknownfunction) CYP39 CYP39A1 CYP46 CYP46A1 CYP51 CYP51A1 (lanosterol 14-alphademethylase)

In some embodiments, a modification at phosphorus results in aP-modification moiety characterized in that it acts as a pro-drug, e.g.,the P-modification moiety facilitates delivery of an oligonucleotide toa desired location prior to removal. For instance, in some embodiments,a P-modification moiety results from PEGylation at the linkagephosphorus. One of skill in the relevant arts will appreciate thatvarious PEG chain lengths are useful and that the selection of chainlength will be determined in part by the result that is sought to beachieved by PEGylation. For instance, in some embodiments, PEGylation iseffected in order to reduce RES uptake and extend in vivo circulationlifetime of an oligonucleotide.

In some embodiments, a PEGylation reagent for use in accordance with thepresent invention is of a molecular weight of about 300 g/mol to about100,000 g/mol. In some embodiments, a PEGylation reagent is of amolecular weight of about 300 g/mol to about 10,000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 300g/mol to about 5,000 g/mol. In some embodiments, a PEGylation reagent isof a molecular weight of about 500 g/mol. In some embodiments, aPEGylation reagent of a molecular weight of about 1000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 3000g/mol. In some embodiments, a PEGylation reagent is of a molecularweight of about 5000 g/mol.

In certain embodiments, a PEGylation reagent is PEG500. In certainembodiments, a PEGylation reagent is PEG1000. In certain embodiments, aPEGylation reagent is PEG3000. In certain embodiments, a PEGylationreagent is PEG5000.

In some embodiments, a P-modification moiety is characterized in that itacts as a PK enhancer, e.g., lipids, PEGylated lipids, etc.

In some embodiments, a P-modification moiety is characterized in that itacts as an agent which promotes cell entry and/or endosomal escape, suchas a membrane-disruptive lipid or peptide.

In some embodiments, a P-modification moiety is characterized in that itacts as a targeting agent. In some embodiments, a P-modification moietyis or comprises a targeting agent. The phrase “targeting agent,” as usedherein, is an entity that is associates with a payload of interest(e.g., with an oligonucleotide or oligonucleotide composition) and alsointeracts with a target site of interest so that the payload of interestis targeted to the target site of interest when associated with thetargeting agent to a materially greater extent than is observed underotherwise comparable conditions when the payload of interest is notassociated with the targeting agent. A targeting agent may be, orcomprise, any of a variety of chemical moieties, including, for example,small molecule moieties, nucleic acids, polypeptides, carbohydrates,etc. Targeting agents are described further by Adarsh et al., “OrganelleSpecific Targeted Drug Delivery—A Review,” International Journal ofResearch in Pharmaceutical and Biomedical Sciences, 2011, p. 895.

Exemplary such targeting agents include, but are not limited to,proteins (e.g. Transferrin), oligopeptides (e.g., cyclic and acylicRGD-containing oligopedptides), antibodies (monoclonal and polyclonalantibodies, e.g. IgG, IgA, IgM, IgD, IgE antibodies), sugars/carbohydrates (e.g., monosaccharides and/or oligosaccharides (mannose,mannose-6-phosphate, galactose, and the like)), vitamins (e.g., folate),or other small biomolecules. In some embodiments, a targeting moiety isa steroid molecule (e.g., bile acids including cholic acid, deoxycholicacid, dehydrocholic acid; cortisone; digoxigenin; testosterone;cholesterol; cationic steroids such as cortisone having atrimethylaminomethyl hydrazide group attached via a double bond at the3-position of the cortisone ring, etc.). In some embodiments, atargeting moiety is a lipophilic molecule (e.g., alicyclic hydrocarbons,saturated and unsaturated fatty acids, waxes, terpenes, andpolyalicyclic hydrocarbons such as adamantine andbuckminsterfullerenes). In some embodiments, a lipophilic molecule is aterpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal.In some embodiments, a targeting moiety is a peptide.

In some embodiments, a P-modification moiety is a targeting agent offormula —X—L—R¹ wherein each of X, L, and R¹ are as defined in Formula Iabove.

In some embodiments, a P-modification moiety is characterized in that itfacilitates cell specific delivery.

In some embodiments, a P-modification moiety is characterized in that itfalls into one or more of the above-described categories. For instance,in some embodiments, a P-modification moiety acts as a PK enhancer and atargeting ligand. In some embodiments, a P-modification moiety acts as apro-drug and an endosomal escape agent. One of skill in the relevantarts would recognize that numerous other such combinations are possibleand are contemplated by the present invention.

Nucleobases

In some embodiments, a nucleobase present in a provided oligonucleotideis a natural nucleobase or a modified nucleobase derived from a naturalnucleobase. Examples include, but are not limited to, uracil, thymine,adenine, cytosine, and guanine having their respective amino groupsprotected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine,5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidineanalogs such as pseudoisocytosine and pseudouracil and other modifiednucleobases such as 8-substituted purines, xanthine, or hypoxanthine(the latter two being the natural degradation products). Exemplarymodified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9,1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7,313.

Compounds represented by the following general formulae are alsocontemplated as modified nucleobases:

wherein R⁸ is an optionally substituted, linear or branched groupselected from aliphatic, aryl, aralkyl, aryloxylalkyl, carbocyclyl,heterocyclyl or heteroaryl group having 1 to 15 carbon atoms, including,by way of example only, a methyl, isopropyl, phenyl, benzyl, orphenoxymethyl group; and each of R⁹ and R¹⁰ is independently anoptionally substituted group selected from linear or branched aliphatic,carbocyclyl, aryl, heterocyclyl and heteroaryl.

Modified nucleobases also include expanded-size nucleobases in which oneor more aryl rings, such as phenyl rings, have been added. Nucleic basereplacements described in the Glen Research catalog(www.glenresearch.com); Krueger A T et al, Acc. Chem. Res., 2007, 40,141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., etal., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr.Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol.,2006, 10, 622-627, are contemplated as useful for the synthesis of thenucleic acids described herein. Some examples of these expanded-sizenucleobases are shown below:

Herein, modified nucleobases also encompass structures that are notconsidered nucleobases but are other moieties such as, but not limitedto, corrin- or porphyrin-derived rings. Porphyrin-derived basereplacements have been described in Morales-Rojas, H and Kool, E T, Org.Lett., 2002, 4, 4377-4380. Shown below is an example of aporphyrin-derived ring which can be used as a base replacement:

In some embodiments, modified nucleobases are of any one of thefollowing structures, optionally substituted:

In some embodiments, a modified nucleobase is fluorescent. Exemplarysuch fluorescent modified nucleobases include phenanthrene, pyrene,stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene,benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uracil,and naphtho-uracil, as shown below:

In some embodiments, a modified nucleobase is unsubstituted. In someembodiments, a modified nucleobase is substituted. In some embodiments,a modified nucleobase is substituted such that it contains, e.g.,heteroatoms, alkyl groups, or linking moieties connected to fluorescentmoieties, biotin or avidin moieties, or other protein or peptides. Insome embodiments, a modified nucleobase is a “universal base” that isnot a nucleobase in the most classical sense, but that functionssimilarly to a nucleobase. One representative example of such auniversal base is 3-nitropyrrole.

In some embodiments, other nucleosides can also be used in the processdisclosed herein and include nucleosides that incorporate modifiednucleobases, or nucleobases covalently bound to modified sugars. Someexamples of nucleosides that incorporate modified nucleobases include4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2′-O-methylcytidine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; dihydrouridine;2′-O-methylpseudouridine; beta,D-galactosylqueosine;2′-O-methylguanosine; N⁶-isopentenyladenosine; 1-methyladenosine;1-methylpseudouridine; 1-methylguanosine; 1-methylinosine;2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine;N⁷-methylguanosine; 3-methyl-cytidine; 5-methylcytidine;5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine;N⁶-methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine;5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine;5-methoxycarbonylmethyluridine; 5-methoxyuridine;2-methylthio-N⁶-isopentenyladenosine;N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;2-thiouridine; 4-thiouridine; 5-methyluridine;2′-O-methyl-5-methyluridine; and 2′-O-methyluridine.

In some embodiments, nucleosides include 6′-modified bicyclic nucleosideanalogs that have either (R) or (S)-chirality at the 6′-position andinclude the analogs described in U.S. Pat. No. 7,399,845. In otherembodiments, nucleosides include 5′-modified bicyclic nucleoside analogsthat have either (R) or (S)-chirality at the 5′-position and include theanalogs described in U.S. Patent Application Publication No.20070287831.

In some embodiments, a nucleobase or modified nucleobase comprises oneor more biomolecule binding moieties such as e.g., antibodies, antibodyfragments, biotin, avidin, streptavidin, receptor ligands, or chelatingmoieties. In other embodiments, a nucleobase or modified nucleobase is5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments,a nucleobase or modified nucleobase is modified by substitution with afluorescent or biomolecule binding moiety. In some embodiments, thesubstituent on a nucleobase or modified nucleobase is a fluorescentmoiety. In some embodiments, the substituent on a nucleobase or modifiednucleobase is biotin or avidin.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200;6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is hereinincorporated by reference in its entirety.

Sugars

The most common naturally occurring nucleotides are comprised of ribosesugars linked to the nucleobases adenosine (A), cytosine (C), guanine(G), and thymine (T) or uracil (U). Also contemplated are modifiednucleotides wherein a phosphate group or linkage phosphorus in thenucleotides can be linked to various positions of a sugar or modifiedsugar. As non-limiting examples, the phosphate group or linkagephosphorus can be linked to the 2′, 3′, 4′ or 5′ hydroxyl moiety of asugar or modified sugar. Nucleotides that incorporate modifiednucleobases as described herein are also contemplated in this context.In some embodiments, nucleotides or modified nucleotides comprising anunprotected —OH moiety are used in accordance with methods of thepresent invention.

Other modified sugars can also be incorporated within a providedoligonucleotide. In some embodiments, a modified sugar contains one ormore substituents at the 2′ position including one of the following: —F;—CF₃, —CN, —N₃, —NO, —NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ isindependently as defined above and described herein; —O—(C₁-C₁₀ alkyl),—S—(C₁-C₁₀ alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀alkeny)^(, —S—(C) ₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀alkenyl)₂; —O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀alkynyl), or —N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)—O—(C₁-C₁₀alkyl), —O—(C₁-C₁₀ alkylene)—NH—(C₁-C₁₀ alkyl) or —O—(C₁C₁₀alkylene)—NH(C₁C₁₀ alkyl)₂, —NH—(C₁-C₁₀ alkylene)—O—(C₁-C₁₀ alkyl), or—N(C₁-C₁₀ alkyl)(C₁C₁₀ alkylene)—O—(C₁-C₁₀ alkyl), wherein the alkyl,alkylene, alkenyl and alkynyl may be substituted or unsubstituted.Examples of substituents include, and are not limited to,—O(CH₂)_(n)OCH₃, and —O(CH₂)_(n)NH₂, wherein n is from 1 to about 10,MOE, DMAOE, DMAEOE. Also contemplated herein are modified sugarsdescribed in WO 2001/088198; and Martin et al., Helv. Chim. Acta, 1995,78, 486-504. In some embodiments, a modified sugar comprises one or moregroups selected from a substituted silyl group, an RNA cleaving group, areporter group, a fluorescent label, an intercalator, a group forimproving the pharmacokinetic properties of a nucleic acid, a group forimproving the pharmacodynamic properties of a nucleic acid, or othersubstituents having similar properties. In some embodiments,modifications are made at one or more of the the 2′, 3′, 4′, 5′, or 6′positions of the sugar or modified sugar, including the 3′ position ofthe sugar on the 3′-terminal nucleotide or in the 5′ position of the5′-terminal nucleotide.

In some embodiments, the 2′-OH of a ribose is replaced with asubstituent including one of the following: —H, —F; —CF₃, —CN, —N₃, —NO,—NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ is independently asdefined above and described herein; —O—(C₁-C₁₀ alkyl), —S—(C₁-C₁₀alkyl), —NH—(C₂-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀ alkenyl),—S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀ alkenyl)₂;—O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀ alkynyl), or—N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)—O—(C₁C₁₀ alkyl), —O—(C₁-C₁₀alkylene)—NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀ alkylene)—NH(C₁-C₁₀ alkyl)₂,—NH—(C₁-C₁₀ alkylene)—O—(C₁-C₁₀ alkyl), —N(C₁-C₁₀ alkyl)—(C₁C₁₀alkylene)—O—(C₁-C₁₀ alkyl), wherein the alkyl, alkylene, alkenyl andalkynyl may be substituted or unsubstituted. In some embodiments, the 2′—OH is replaced with H (deoxyribose). In some embodiments, the 2′ —OH isreplaced with —F. In some embodiments, the 2′ —OH is replaced with —OR′.In some embodiments, the 2′ —OH is replaced with —OMe. In someembodiments, the 2′ —OH is replaced with —OCH₂CH₂OMe.

Modified sugars also include locked nucleic acids (LNAs). In someembodiments, two substituents on sugar carbon atoms are taken togetherto form a bivalent moiety. In some embodiments, two substituents are ontwo different sugar carbon atoms. In some embodiments, a formed bivalentmoiety has the structure of L as defined herein. In some embodiments,—L— is —O—CH₂, wherein —CH₂— is optionally substituted. In someembodiments, —L— is —O—CH₂. In some embodiments, —L— is —O—CH(Et)—. Insome embodiments, —L— is between C2 and C4 of a sugar moiety. In someembodiments, a locked nucleic acid has the structure indicated below. Alocked nucleic acid of the structure below is indicated, wherein Barepresents a nucleobase or modified nucleobase as described herein, andwherein R^(2s) is —OCH₂C4′.

In some embodiments, a modified sugar is an ENA such as those describedin, e.g., Seth et al., J Am Chem Soc. 2010 October 27; 132(42):14942-14950. In some embodiments, a modified sugar is any of those foundin an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol,threose, 2′ fluoroarabinose, or cyclohexene.

Modified sugars include sugar mimetics such as cyclobutyl or cyclopentylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;5,118,800; 5,319,080 ; and 5,359,044. Some modified sugars that arecontemplated include sugars in which the oxygen atom within the ribosering is replaced by nitrogen, sulfur, selenium, or carbon. In someembodiments, a modified sugar is a modified ribose wherein the oxygenatom within the ribose ring is replaced with nitrogen, and wherein thenitrogen is optionally substituted with an alkyl group (e.g., methyl,ethyl, isopropyl, etc).

Non-limiting examples of modified sugars include glycerol, which formglycerol nucleic acid (GNA) analogues. One example of a GNA analogue isshown below and is described in Zhang, R et al., J. Am. Chem. Soc.,2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc., 2005, 127,4174-4175 and Tsai CH et al., PNAS, 2007, 14598-14603 (X═O⁻):

Another example of a GNA derived analogue, flexible nucleic acid (FNA)based on the mixed acetal aminal of formyl glycerol, is described inJoyce GF et al., PNAS, 1987, 84, 4398-4402 and Heuberger BD and SwitzerC, J. Am. Chem. Soc., 2008, 130, 412-413, and is shown below:

Additional non-limiting examples of modified sugars includehexopyranosyl (6′ to 4′), pentopyranosyl (4′ to 2′), pentopyranosyl (4′to 3′), or tetrofuranosyl (3′ to 2′) sugars. In some embodiments, ahexopyranosyl (6′ to 4′) sugar is of any one in the following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 2′) sugar is of any one inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 3′) sugar is of any one inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a tetrofuranosyl (3′ to 2′) sugar is of either inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a modified sugar is of any one in the followingformulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, one or more hydroxyl group in a sugar moiety isoptionally and independently replaced with halogen, R′ —N(R′)₂, —OR′, or—SR′, wherein each R′ is independently as defined above and describedherein.

In some embodiments, a sugar mimetic is as illustrated below, whereinX^(s) corresponds to the P-modification group “—XLR¹” described herein,Ba is as defined herein, and X¹ is selected from —S—, —Se—, —CH₂, —NMe—,—NEt— or —NiPr—.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more(e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more), inclusive,of the sugars in a chirally controlled oligonucleotide composition aremodified. In some embodiments, only purine residues are modified (e.g.,about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, , 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more] of the purine residues are modified). Insome embodiments, only pyrimidine residues are modified (e.g., about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more] of the pyridimine residues are modified). In someembodiments, both purine and pyrimidine residues are modified.

Modified sugars and sugar mimetics can be prepared by methods known inthe art, including, but not limited to: A. Eschenmoser, Science (1999),284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M.Egli et al, J. Am. Chem. Soc. (2006), 128(33):10847-56; A. Eschenmoserin Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V.Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p.293; K. -U.Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al,Helv. Chim. Acta (1992), 75:218; J. Hunziker et al, Helv. Chim. Acta(1993), 76:259; G. Otting et al, Hely. Chim. Acta (1993), 76:2701; K.Groebke et al, Hely. Chim. Acta (1998), 81:375; and A. Eschenmoser,Science (1999), 284:2118. Modifications to the 2′ modifications can befound in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and allreferences therein. Specific modifications to the ribose can be found inthe following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem.,1993, 36, 831- 841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79,1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310). Insome embodiments, a modified sugar is any of those described in PCTPublication No. W02012/030683, incorporated herein by reference, anddepicted in the FIGS. 26-30 of the present application.

Oligonucleotides

In some embodiments, the present invention provides oligonucleotides andoligonucleotide compositions that are chirally controlled. For instance,in some embodiments, a provided composition contains predeterminedlevels of one or more individual oligonucleotide types, wherein anoligonucleotide type is defined by: 1) base sequence; 2) pattern ofbackbone linkages; 3) pattern of backbone chiral centers; and 4) patternof backbone P-modifications.

In some embodiments, a provided oligonucleotide is a unimer. In someembodiments, a provided oligonucleotide is a P-modification unimer. Insome embodiments, a provided oligonucleotide is a stereounimer. In someembodiments, a provided oligonucleotide is a stereounimer ofconfiguration Rp. In some embodiments, a provided oligonucleotide is astereounimer of configuration Sp.

In some embodiments, a provided oligonucleotide is an altmer. In someembodiments, a provided oligonucleotide is a P-modification altmer. Insome embodiments, a provided oligonucleotide is a stereoaltmer.

In some embodiments, a provided oligonucleotide is a blockmer. In someembodiments, a provided oligonucleotide is a P-modification blockmer. Insome embodiments, a provided oligonucleotide is a stereoblockmer.

In some embodiments, a provided oligonucleotide is a gapmer.

In some embodiments, a provided oligonucleotide is a skipmer.

In some embodiments, a provided oligonucleotide is a hemimer. In someembodiments, a hemimer is an oligonucleotide wherein the 5′-end or the3′-end has a sequence that possesses a structure feature that the restof the oligonucleotide does not have. In some embodiments, the 5′-end orthe 3′-end has or comprises 2 to 20 nucleotides. In some embodiments, astructural feature is a base modification. In some embodiments, astructural feature is a sugar modification. In some embodiments, astructural feature is a P-modification. In some embodiments, astructural feature is stereochemistry of the chiral internucleotidiclinkage. In some embodiments, a structural feature is or comprises abase modification, a sugar modification, a P-modification, orstereochemistry of the chiral internucleotidic linkage, or combinationsthereof. In some embodiments, a hemimer is an oligonucleotide in whicheach sugar moiety of the 5′-end sequence shares a common modification.In some embodiments, a hemimer is an oligonucleotide in which each sugarmoiety of the 3′-end sequence shares a common modification. In someembodiments, a common sugar modification of the 5′ or 3′ end sequence isnot shared by any other sugar moieties in the oligonucleotide. In someembodiments, an exemplary hemimer is an oligonucleotide comprising asequence of substituted or unsubstituted 2′-O-alkyl sugar modifiednucleosides, bicyclic sugar modified nucleosides, β-D-ribonucleosides orβ-D- deoxyribonucleosides (for example 2′-MOE modified nucleosides, andLNA™ or ENA™ bicyclic syugar modified nucleosides) at one terminus and asequence of nucleosides with a different sugar moiety (such as asubstituted or unsubstituted 2′-O-alkyl sugar modified nucleosides,bicyclic sugar modified nucleosides or natural ones) at the otherterminus. In some embodiments, a provided oligonucleotide is acombination of one or more of unimer, altmer, blockmer, gapmer, hemimerand skipmer. In some embodiments, a provided oligonucleotide is acombination of one or more of unimer, altmer, blockmer, gapmer, andskipmer. For instance, in some embodiments, a provided oligonucleotideis both an altmer and a gapmer. In some embodiments, a providednucleotide is both a gapmer and a skipmer. One of skill in the chemicaland synthetic arts will recognize that numerous other combinations ofpatterns are available and are limited only by the commercialavailability and/or synthetic accessibility of constituent partsrequired to synthesize a provided oligonucleotide in accordance withmethods of the present invention. In some embodiments, a hemimerstructure provides advantageous benefits, as exemplified by FIG. 29. Insome embodiments, provided oligonucleotides are 5′-hemmimers thatcomprises modified sugar moieties in a 5′-end sequence. In someembodiments, provided oligonucleotides are 5′-hemmimers that comprisesmodified 2′-sugar moieties in a 5′-end sequence.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleotides. In some embodiments, a providedoligonucleotide comprises one or more modified nucleotides. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted nucleosides. In some embodiments, a provided oligonucleotidecomprises one or more modified nucleosides. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedLNAs.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleobases. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted naturalnucleobases. In some embodiments, a provided oligonucleotide comprisesone or more optionally substituted modified nucleobases. In someembodiments, a provided oligonucleotide comprises one or more5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or5-carboxylcytosine. In some embodiments, a provided oligonucleotidecomprises one or more 5-methylcytidine.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted sugars. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted sugarsfound in naturally occurring DNA and RNA. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedribose or deoxyribose. In some embodiments, a provided oligonucleotidecomprises one or more optionally substituted ribose or deoxyribose,wherein one or more hydroxyl groups of the ribose or deoxyribose moietyis optionally and independently replaced by halogen, R′, —N(R′)₂, —OR′,or —SR′, wherein each R′ is independently as defined above and describedherein. In some embodiments, a provided oligonucleotide comprises one ormore optionally substituted deoxyribose, wherein the 2′ position of thedeoxyribose is optionally and independently substituted with halogen,R′, —N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as definedabove and described herein. In some embodiments, a providedoligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with halogen. In some embodiments, aprovided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with one or more —F. halogen. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently as defined above and described herein. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ aliphatic. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ alkyl. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OMe. In some embodiments,a provided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with —O—methoxyethyl.

In some embodiments, a provided oligonucleotide is single-strandedoligonucleotide.

In some embodiments, a provided oligonucleotide is a hybridizedoligonucleotide strand. In certain embodiments, a providedoligonucleotide is a partially hydridized oligonucleotide strand. Incertain embodiments, a provided oligonucleotide is a completelyhydridized oligonucleotide strand. In certain embodiments, a providedoligonucleotide is a double-stranded oligonucleotide. In certainembodiments, a provided oligonucleotide is a triple-strandedoligonucleotide (e.g., a triplex).

In some embodiments, a provided oligonucleotide is chimeric. Forexample, in some embodiments, a provided oligonucleotide is DNA-RNAchimera, DNA-LNA chimera, etc.

In some embodiments, any one of the structures comprising anoligonucleotide depicted in WO2012/030683 can be modified in accordancewith methods of the present invention to provide chirally controlledvariants thereof. For example, in some embodiments the chirallycontrolled variants comprise a stereochemical modification at any one ormore of the linkage phosphorus and/or a P-modification at any one ormore of the linkage phosphorus. For example, in some embodiments, aparticular nucleotide unit of a oligonucleotide of WO2012/030683 ispreselected to be stereochemically modified at the linkage phosphorus ofthat nucleotide unit and/or P-modified at the linkage phosphorus of thatnucleotide unit. In some embodiments, a chirally controlledoligonucleotide is of any one of the structures depicted in FIGS. 26-30.In some embodiments, a chirally controlled oligonucleotide is a variant(e.g., modified version) of any one of the structures depicted in FIGS.26-30. The disclosure of WO2012/030683 is herein incorporated byreference in its entirety.

In some embodiments, a provided oligonucleotide is a therapeutic agent.

In some embodiments, a provided oligonucleotide is an antisenseoligonucleotide.

In some embodiments, a provided oligonucleotide is an antigeneoligonucleotide.

In some embodiments, a provided oligonucleotide is a decoyoligonucleotide.

In some embodiments, a provided oligonucleotide is part of a DNAvaccine.

In some embodiments, a provided oligonucleotide is an immunomodulatoryoligonucleotide, e.g., immunostimulatory oligonucleotide andimmunoinhibitory oligonucleotide.

In some embodiments, a provided oligonucleotide is an adjuvant.

In some embodiments, a provided oligonucleotide is an aptamer.

In some embodiments, a provided oligonucleotide is a ribozyme.

In some embodiments, a provided oligonucleotide is a deoxyribozyme(DNAzymes or DNA enzymes).

In some embodiments, a provided oligonucleotide is an siRNA.

In some embodiments, a provided oligonucleotide is a microRNA, or miRNA.

In some embodiments, a provided oligonucleotide is a ncRNA (non-codingRNAs), including a long non-coding RNA (lncRNA) and a small non-codingRNA, such as piwi- interacting RNA (piRNA).

In some embodiments, a provided oligonucleotide is complementary to astructural RNA, e.g., tRNA.

In some embodiments, a provided oligonucleotide is a nucleic acidanalog, e.g., GNA, LNA, PNA, TNA and Morpholino.

In some embodiments, a provided oligonucleotide is a P-modified prodrug.

In some embodiments, a provided oligonucleotide is a primer. In someembodiments, a primers is for use in polymerase-based chain reactions(i.e., PCR) to amplify nucleic acids. In some embodiments, a primer isfor use in any known variations of PCR, such as reverse transcriptionPCR (RT-PCR) and real-time PCR.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to modulate RNase H activation. For example, in someembodiments, RNase H activation is modulated by the presence ofstereocontrolled phosphorothioate nucleic acid analogs, with naturalDNA/RNA being more or equally susceptible than the Rp stereoisomer,which in turn is more susceptible than the corresponding Spstereoisomer.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to indirectly or directly increase or decreaseactivity of a protein or inhibition or promotion of the expression of aprotein. In some embodiments, a provided oligonucleotide ischaracterized in that it is useful in the control of cell proliferation,viral replication, and/or any other cell signaling process.

In some embodiments, a provided oligonucleotide is from about 2 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 4 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 5 to about10 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 10 to about 30 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 15 toabout 25 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.

In some embodiments, the oligonucleotide is at least 2 nucleotide unitsin length. In some embodiments, the oligonucleotide is at least 3nucleotide units in length. In some embodiments, the oligonucleotide isat least 4 nucleotide units in length. In some embodiments, theoligonucleotide is at least 5 nucleotide units in length. In someembodiments, the oligonucleotide is at least 6 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 7nucleotide units in length. In some embodiments, the oligonucleotide isat least 8 nucleotide units in length. In some embodiments, theoligonucleotide is at least 9 nucleotide units in length. In someembodiments, the oligonucleotide is at least 10 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 11nucleotide units in length. In some embodiments, the oligonucleotide isat least 12 nucleotide units in length. In some embodiments, theoligonucleotide is at least 13 nucleotide units in length. In someembodiments, the oligonucleotide is at least 14 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 15nucleotide units in length. In some embodiments, the oligonucleotide isat least 16 nucleotide units in length. In some embodiments, theoligonucleotide is at least 17 nucleotide units in length. In someembodiments, the oligonucleotide is at least 18 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 19nucleotide units in length. In some embodiments, the oligonucleotide isat least 20 nucleotide units in length. In some embodiments, theoligonucleotide is at least 21 nucleotide units in length. In someembodiments, the oligonucleotide is at least 22 nucleotide units inlength. In some embodiments, the oligonucleotide is at least 23nucleotide units in length. In some embodiments, the oligonucleotide isat least 24 nucleotide units in length. In some embodiments, theoligonucleotide is at least 25 nucleotide units in length. In some otherembodiments, the oligonucleotide is at least 30 nucleotide units inlength. In some other embodiments, the oligonucleotide is a duplex ofcomplementary strands of at least 18 nucleotide units in length. In someother embodiments, the oligonucleotide is a duplex of complementarystrands of at least 21 nucleotide units in length.

In some embodiments, the 5′-end and/or the 3′-end of a providedoligonucleotide is modified. In some embodiments, the 5′-end and/or the3′-end of a provided oligonucleotide is modified with a terminal capmoiety. Exemplary such modifications, including terminal cap moietiesare extensively described herein and in the art, for example but notlimited to those described in US Patent Application Publication US2009/0023675A1.

In some embodiments, oligonucleotides of an oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        have the same chemical structure. For example, they have the        same base sequence, the same pattern of backbone linkages (i.e.,        pattern of internucleotidic linkage types, for example,        phosphate, phosphorothioate, etc), the same pattern of backbone        chiral centers (i.e. pattern of linkage phosphorus        stereochemistry (Rp/Sp)), and the same pattern of backbone        phosphorus modifications (e.g., pattern of “—XLR¹” groups in        formula I).        Species of Oligonucleotides

In some embodiments, a provided chirally controlled oligonucleotidecomprises the sequence of, or part of the sequence of mipomersen.Mipomersen is based on the following base sequenceGCCT/UCAGT/UCT/UGCT/UT/UCGCACC (SEQ ID NO: 64). In some embodiments, oneor more of any of the nucleotide or linkages may be modified inaccordance of the present invention. In some embodiments, the presentinvention provides a chirally controlled oligonucleotide having thesequence ofG*—C*—C*—U*—C*—dA—dG—dT—dC—dT—dG—dmC—dT—dT—dmC—G*—C*—A*—C*—C* (SEQ IDNO: 65) [d=2′-deoxy, *=2′-O-(2-methoxyethyl)] with 3′→5′phosphorothioate linkages. Exemplary modified mipomersen sequences aredescribed throughout the application, including but not limited to thosein Table 2.

In certain embodiments, a provided oligonucleotide is a mipomersenunimer. In certain embodiments, a provided oligonucleotide is amipomersen unimer of configuration Rp. In certain embodiments, aprovided oligonucleotide is a mipomersen unimer of configuration Sp.

Exempary chirally controlled oligonucleotides comprising the sequenceof, or part of the sequence of mipomersen is depicted in Table 2, below.

TABLE 2  Exemplary Mipomersen related sequences. SEQ ID OligoStereochemistry/Sequence Description NO: 101 All-(Rp)- All-R  66d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 102 All-(Sp)- All-S  67d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 103(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, 5R-9S-5R  68Rp, Rp, Rp, Rp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 104(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-5S  69Sp, Sp, Sp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 105(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, 1S-17R-1S  70Rp, Rp, Rp, Rp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 106(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 1R-17S-1R 71 Sp, Sp, Sp, Rp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 107(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, (R/S)₉R  72Sp, Rp, Sp, Rp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 108(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, (S/R)₉S  73Rp, Sp, Rp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 109(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, 3S-13R-3S  74Rp, Rp, Sp, Sp, Sp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 110(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 3R-13S-3R 75 Sp, Rp, Rp, Rp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 111(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 18S/R¹⁹  76Sp, Sp, Sp, Rp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 112(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, 18S/R⁹  77Sp, Sp, Sp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 113(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 18S/R²  78Sp, Sp, Sp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 114(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, (RRS)₆-R  79Sp, Rp, Rp, Sp, Rp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 115(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, S-(RRS)₆  80Rp, Sp, Rp, Rp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 116(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR  81Rp, Rp, Sp, Rp Rp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 122All-(Rp)- All-R  82d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1 As1Cs1C] 123(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, 1S-17R-1S  83Rp, Rp, Rp, Rp, Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] 124All-(Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1 All-S  84Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] 126 All-(Rp)-d[Cs2As2Gs2T] All-R 127All-(Rp)-d[Cs3As3Gs3T] All-R 128 All-(Sp)-d[Cs4As4Gs4T] All-S 129All-(Sp)-d[Cs5As5Gs5T] All-S 130 All-(Sp)-d[Cs6As6Gs6T] All-S 131All-(Rp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7 All-R  85Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] 132All-(Sp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7 All-S  86Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] 133(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, 5R-9S-5R  87Rp, Rp, Rp, Rp)- d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 134(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-5S  88Sp, Sp, Sp, Sp)- d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 135All-(Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-R  89 136All-(Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-S  90 137(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp)- 1S-9R-1S  91d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 138(Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp)- 2S-7R-2S  92d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 139(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp)- 1R-9S-1R  93d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 140(Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp)- 2R-7S-2R  94d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 141(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp)- 3S-5R-3S  95d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 142(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp)- 3R-5S-3R  96d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 143(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp)- (SSR)₃-SS  97d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 144(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp)- (RRS)₃-RR  98d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 145 All-(Rp)- All-R  99d[5mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1 Gs15mC] 146All-(Rp)-d[Gs15mCs1Ts1G] All-R 147 All-(Rp)-d[5mCs1As1Gs1T] All-R 148All-(Rp)-d[5mCs2As2Gs2Ts25mCs2Ts2Gs25mCs2Ts2Ts25mCs2G] All-R 100 149All-(Rp)-d[5mCs4As4Gs4Ts45mCs4Ts4Gs45mCs4Ts4Ts45mCs4G] All-R 101 151All-(Sp)-d[Cs1AsGs1T] All-S 152 All-(Sp)-d[Cs1AGs1T] All-S 153All-(Sp)-d[CAs1GsT] All-S 157 All-(Sp)-d[5mCs1As1Gs1T] All-S 158(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 102Sp, Sp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCs1GsCsACsC] 159(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-5S 103Sp, Sp, Sp, Sp)- d[Gs1Cs1Cs1Ts1CsAsGsTsCsTsGsCsTsTsCs1GsCs2As2Cs2C] 160All-(Rp)- All-R 104(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 161 All-(Sp)- All-S 105(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 162(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, 5R-9S-5R 106Rp, Rp, Rp, Rp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 163(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-5S 107Sp, Sp, Sp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 164(Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, 1S-17R-1S 108Rp, Rp, Rp, Rp, Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 165(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 1R-17S-1R109 Sp, Sp, Sp, Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 166(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, (R/S)₉R 110Sp, Rp, Sp, Rp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 167(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, (S/R)₉S 111Rp, Sp, Rp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 168(Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, 3S-13R-3S 112Rp, Rp, Sp, Sp, Sp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 169(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 3R-13S-3R113 Sp, Rp, Rp, Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 170(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 18S/R¹⁹ 114Sp, Sp, Sp, Rp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] (Gs5mCsAs5mCs5mC)_(MOE) 171(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, 18S/R⁹ 115Sp, Sp, Sp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 172(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, 18S/R² 116Sp, Sp, Sp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 173(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, (RRS)₆-R 117Sp, Rp, Rp, Sp, Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 174(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, S-(RRS)₆ 118Rp, Sp, Rp, Rp, Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 175(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 119Rp, Rp, Sp, Rp Rp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 176(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 120Rp, Rp, Sp, Rp Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1] (Gs15mCs1As15mCs15mC)_(MOE) 177(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 121Rp, Rp, Sp, Rp Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[AGT5mCTG5mCTT5mC](Gs25mCs2As25mCs25mC)_(MOE) 178(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, S-(RRS)₆ 122Rp, Sp, Rp, Rp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(F) (F: 2-fluorodeoxyribose) 179(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 123Rp, Rp, Sp, Rp Rp)d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs8As8Cs8C] 180(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 124Rp, Rp, Sp, Rp Rp)d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs9As9Cs9C] 181(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 125Rp, Rp, Sp, Rp Rp)d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10Ts10Cs10Gs10Cs10As10Cs10C] 182(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 126Rp, Rp, Sp, Rp Rp)d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11Ts11Cs11Gs11Cs11As11Cs11C] 183(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 127Rp, Rp, Sp, Rp Rp)d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12Ts12Cs12Gs12Cs12As12Cs12C] 184(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 128Rp, Rp, Sp, Rp Rp)d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13Ts13Cs13Gs13Cs13As13Cs13C] 185(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 129Rp, Rp, Sp, Rp Rp)d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14Ts14Cs14Gs14Cs14As14Cs14C] 186(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 130Rp, Rp, Sp, Rp Rp)d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15Ts15Cs15Gs15Cs15As15Cs15C] 187(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, RS-(RRS)₅-RR 131Rp, Rp, Sp, Rp Rp)d[GsCsCs1TsCsAs]GsUs2CsUsGsd[CsTs3TsCsGs]CsAs4CsC 188(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 132Sp, Sp, Sp)- d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsACsC] 189(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 133Sp, Sp, Sp)- d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1CsACs1C]190 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S134 Sp, Sp, Sp)-d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs1ACs8C] 191(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 135Sp, Sp, Sp)- d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs1ACs9C]192 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S136 Sp, Sp, Sp)- d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10Ts10Cs10Gs10Cs1ACs10C] 193(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 137Sp, Sp, Sp)- d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11Ts11Cs11Gs11Cs1ACs11C] 194(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 138Sp, Sp, Sp)- d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12Ts12Cs12Gs12Cs1ACs12C] 195(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 139Sp, Sp, Sp)- d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13Ts13Cs13Gs13Cs1ACs13C] 196(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 140Sp, Sp, Sp)- d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14Ts14Cs14Gs14Cs1ACs14C] 197(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 141Sp, Sp, Sp)- d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15Ts15Cs15Gs15Cs1ACs15C] 198(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 142Sp, Sp, Sp)- GsCsCsUsCsAsGsUsCsUsGsCsUsUsCsGsCsACsC 199(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 143Sp, Sp, Sp)- Gs1Cs1Cs1Us1Cs1As1Gs1Us1Cs1Us1Gs1Cs1Us1Us1Cs1Gs1CsACs1C 200(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 144Sp, Sp, Sp)- Gs8Cs8Cs8Us8Cs8As8Gs8Us8Cs8Us8Gs8Cs8Us8Us8Cs8Gs8Cs1ACs8C201 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S145 Sp, Sp, Sp)-Gs9Cs9Cs9Us9Cs9As9Gs9Us9Cs9Us9Gs9Cs9Us9Us9Cs9Gs9Cs1ACs9C 202(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 146Sp, Sp, Sp)- Gs10Cs10Cs10Us10Cs10As10Gs10Us10Cs10Us10Gs10Cs10Us10Us10Cs10Gs10Cs1ACs10C 203(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 147Sp, Sp, Sp)- Gs11Cs11Cs11Us11Cs11As11Gs11Us11Cs11Us11Gs11Cs11Us11Us11Cs11Gs11Cs1ACs11C 204(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 148Sp, Sp, Sp)- Gs12Cs12Cs12Us12Cs12As12Gs12Us12Cs12Us12Gs12Cs12Us12Us12Cs12Gs12Cs1ACs12C 205(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 149Sp, Sp, Sp)- Gs13Cs13Cs13Us13Cs13As13Gs13Us13Cs13Us13Gs13Cs13Us13Us13Cs13Gs13Cs1ACs13C 206(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 150Sp, Sp, Sp)- Gs14Cs14Cs14Us14Cs14As14Gs14Us14Cs14Us14Gs14Cs14Us14Us14Cs14Gs14Cs1ACs14C 207(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, 5S-9R-4S 151Sp, Sp, Sp)- Gs15Cs15Cs15Us15Cs15As15Gs15Us15Cs15Us15Gs15Cs15Us15Us15Cs15Gs15Cs1ACs15COligonucleotide Compositions

The present invention provides compositions comprising or consisting ofa plurality of provided oligonucleotides (e.g., chirally controlledoligonucleotide compositions). In some embodiments, all such providedoligonucleotides are of the same type, i.e., all have the same basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “—XLR¹” groups in formula I).In many embodiments, however, provided compositions comprise a pluralityof oligonucleotides types, typically in pre-determined relative amounts.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a chirally pure mipomersen composition. That is to say,in some embodiments, a provided chirally controlled oligonucleotidecomposition provides mipomersen as a single diastereomer with respect tothe configuration of the linkage phosphorus.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a chirally uniform mipomersen composition. That is tosay, in some embodiments, every linkage phosphorus of mipomersen is inthe Rp configuration or every linkage phosphorus of mipomersen is in theSp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of one or more providedoligonucleotide types. One of skill in the chemical and medicinal artswill recognize that the selection and amount of each of the one or moretypes of provided oligonucleotides in a provided composition will dependon the intended use of that composition. That is to say, one of skill inthe relevant arts would design a provided chirally controlledoligonucleotide composition such that the amounts and types of providedoligonucleotides contained therein cause the composition as a whole tohave certain desirable characteristics (e.g., biologically desirable,therapeutically desirable, etc.).

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of two or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of threeor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof four or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of five or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of six or more provided oligonucleotide types.In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of seven or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of eightor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof nine or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of ten or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of fifteen or more provided oligonucleotidetypes.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration and an amount of chirally uniform mipomersen ofthe Sp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration, an amount of chirally uniform mipomersen of theSp configuration, and an amount of one or more chirally pure mipomersenof a desired diastereomeric form.

In some embodiments, a provided oligonucleotide type is selected fromthose described in PCT/US2013/050407, which is incorporated herein byreference. In some embodiments, a provided chirally controlledoligonucleotide composition comprises oligonucleotides of aoligonucleotide type selected from those described in PCT/US2013/050407.

Methods for Making Chirally Controlled Oligonucleotides and CompositionsThereof

The present invention provides methods for making chirally controlledoligonucleotides and chirally controlled compositions comprising one ormore specific nucleotide types. As noted above, the phrase“oligonucleotide type,” as used herein, defines an oligonucleotide thathas a particular base sequence, pattern of backbone linkages, pattern ofbackbone chiral centers, and pattern of backbone phosphorusmodifications (e.g., “—XLR¹” groups). Oligonucleotides of a commondesignated “type” are structurally identical to one another with respectto base sequence, pattern of backbone linkages, pattern of backbonechiral centers, and pattern of backbone phosphorus modifications.

In some embodiments, a provided chirally controlled oligonucleotide inthe invention has properties different from those of the correspondingstereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has lipophilicity different from that of thestereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has different retention time on HPLC. In someembodiments, a chirally controlled oligonucleotide may have a peakretention time significantly different from that of the correspondingstereorandom oligonucleotide mixture. During oligonucleotidepurification using HPLC as generally practiced in the art, certainchirally controlled oligonucleotides will be largely if not totallylost. During oligonucleotide purification using HPLC as generallypracticed in the art, certain chirally controlled oligonucleotides willbe largely if not totally lost. One of the consequences is that certaindiastereomers of a stereorandom oligonucleotide mixture (certainchirally controlled oligonucleotides) are not tested in assays. Anotherconsequence is that from batches to batches, due to the inevitableinstrumental and human errors, the supposedly “pure” stereorandomoligonucleotide will have inconsistent compositions in thatdiastereomers in the composition, and their relative and absoluteamounts, are different from batches to batches. The chirally controlledoligonucleotide and chirally controlled oligonucleotide compositionprovided in this invention overcome such problems, as a chirallycontrolled oligonucleotide is synthesized in a chirally controlledfashion as a single diastereomer, and a chirally controlledoligonucleotide composition comprise predetermined levels of one or moreindividual oligonucleotide types.

One of skill in the chemical and synthetic arts will appreciate thatsynthetic methods of the present invention provide for a degree ofcontrol during each step of the synthesis of a provided oligonucleotidesuch that each nucleotide unit of the oligonucleotide can be designedand/or selected in advance to have a particular stereochemistry at thelinkage phosphorus and/or a particular modification at the linkagephosphorus, and/or a particular base, and/or a particular sugar. In someembodiments, a provided oligonucleotide is designed and/or selected inadvance to have a particular combination of stereocenters at the linkagephosphorus of the internucleotidic linkage.

In some embodiments, a provided oligonucleotide made using methods ofthe present invention is designed and/or determined to have a particularcombination of linkage phosphorus modifications. In some embodiments, aprovided oligonucleotide made using methods of the present invention isdesigned and/or determined to have a particular combination of bases. Insome embodiments, a provided oligonucleotide made using methods of thepresent invention is designed and/or determined to have a particularcombination of sugars. In some embodiments, a provided oligonucleotidemade using methods of the present invention is designed and/ordetermined to have a particular combination of one or more of the abovestructural characteristics.

Methods of the present invention exhibit a high degree of chiralcontrol. For instance, methods of the present invention facilitatecontrol of the stereochemical configuration of every single linkagephosphorus within a provided oligonucleotide. In some embodiments,methods of the present invention provide an oligonucleotide comprisingone or more modified internucleotidic linkages independently having thestructure of formula I.

In some embodiments, methods of the present invention provide anoligonucleotide which is a mipomersen unimer. In some embodiments,methods of the present invention provide an oligonucleotide which is amipomersen unimer of configuration Rp. In some embodiments, methods ofthe present invention provide an oligonucleotide which is a mipomersenunimer of configuration Sp.

In some embodiments, methods of the present invention provide a chirallycontrolled oligonucleotide composition, i.e., an oligonucleotidecomposition that contains predetermined levels of individualoligonucleotide types. In some embodiments a chirally controlledoligonucleotide composition comprises one oligonucleotide type. In someembodiments, a chirally controlled oligonucleotide composition comprisesmore than one oligonucleotide type. In some embodiments, a chirallycontrolled oligonucleotide composition comprises a plurality ofoligonucleotide types. Exemplary chirally controlled oligonucleotidecompositions made in accordance with the present invention are describedherein.

In some embodiments, methods of the present invention provide chirallypure mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent invention provide compositions of mipomersen wherein mipomersenexists in the composition in the form of a single diastereomer withrespect to the configuration of the linkage phosphorus.

In some embodiments, methods of the present invention provide chirallyuniform mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent invention provide compositions of mipomersen in which allnucleotide units therein have the same stereochemistry with respect tothe configuration of the linkage phosphorus, e.g., all nucleotide unitsare of the Rp configuration at the linkage phosphorus or all nucleotideunits are of the Sp configuration at the linkage phosphorus.

In some embodiments, a provided chirally controlled oligonucleotide isover 50% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 55% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 60% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 65% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 70% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 75% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 80% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 85% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 90% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 91% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 92% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 93% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 94% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 95% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 96% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 97% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 98% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 99% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 99.5% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 99.6% pure. In some embodiments, aprovided chirally controlled oligonucleotide is over about 99.7% pure.In some embodiments, a provided chirally controlled oligonucleotide isover about 99.8% pure. In some embodiments, a provided chirallycontrolled oligonucleotide is over about 99.9% pure. In someembodiments, a provided chirally controlled oligonucleotide is over atleast about 99% pure.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise a single oligonucleotide type. Incertain embodiments, such compositions are about 50% diastereomericallypure. In some embodiments, such compositions are about 50%diastereomerically pure. In some embodiments, such compositions areabout 50% diastereomerically pure. In some embodiments, suchcompositions are about 55% diastereomerically pure. In some embodiments,such compositions are about 60% diastereomerically pure. In someembodiments, such compositions are about 65% diastereomerically pure. Insome embodiments, such compositions are about 70% diastereomericallypure. In some embodiments, such compositions are about 75%diastereomerically pure. In some embodiments, such compositions areabout 80% diastereomerically pure. In some embodiments, suchcompositions are about 85% diastereomerically pure. In some embodiments,such compositions are about 90% diastereomerically pure. In someembodiments, such compositions are about 91% diastereomerically pure. Insome embodiments, such compositions are about 92% diastereomericallypure. In some embodiments, such compositions are about 93%diastereomerically pure. In some embodiments, such compositions areabout 94% diastereomerically pure. In some embodiments, suchcompositions are about 95% diastereomerically pure. In some embodiments,such compositions are about 96% diastereomerically pure. In someembodiments, such compositions are about 97% diastereomerically pure. Insome embodiments, such compositions are about 98% diastereomericallypure. In some embodiments, such compositions are about 99%diastereomerically pure. In some embodiments, such compositions areabout 99.5% diastereomerically pure. In some embodiments, suchcompositions are about 99.6% diastereomerically pure. In someembodiments, such compositions are about 99.7% diastereomerically pure.In some embodiments, such compositions are about 99.8%diastereomerically pure. In some embodiments, such compositions areabout 99.9% diastereomerically pure. In some embodiments, suchcompositions are at least about 99% diastereomerically pure.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise multiple oligonucleotide types. Insome embodiments, methods of the present invention allow for thegeneration of a library of chirally controlled oligonucleotides suchthat a pre-selected amount of any one or more chirally controlledoligonucleotide types can be mixed with any one or more other chirallycontrolled oligonucleotide types to create a chirally controlledoligonucleotide composition. In some embodiments, the pre-selectedamount of an oligonucleotide type is a composition having any one of theabove-described diastereomeric purities.

In some embodiments, the present invention provides methods for making achirally controlled oligonucleotide comprising steps of:

-   -   (1) coupling;    -   (2) capping;    -   (3) modifying;    -   (4) deblocking; and    -   (5) repeating steps (1)-(4) until a desired length is achieved.

When describing the provided methods, the word “cycle” has its ordinarymeaning as understood by a person of ordinary skill in the art. In someembodiments, one round of steps (1)-(4) is referred to as a cycle.

In some embodiments, the present invention provides methods for makingchirally controlled oligonucleotide compositions, comprising steps of:

-   -   (a) providing an amount of a first chirally controlled        oligonucleotide; and    -   (b) optionally providing an amount of one or more additional        chirally controlled oligonucleotides.

In some embodiments, a first chirally controlled oligonucleotide is anoligonucleotide type, as described herein. In some embodiments, a one ormore additional chirally controlled oligonucleotide is a one or moreoligonucleotide type, as described herein.

One of skill in the relevant chemical and synthetic arts will recognizethe degree of versatility and control over structural variation andstereochemical configuration of a provided oligonucleotide whensynthesized using methods of the present invention. For instance, aftera first cycle is complete, a subsequent cycle can be performed using anucleotide unit individually selected for that subsequent cycle which,in some embodiments, comprises a nucleobase and/or a sugar that isdifferent from the first cycle nucleobase and/or sugar. Likewise, thechiral auxiliary used in the coupling step of the subsequent cycle canbe different from the chiral auxiliary used in the first cycle, suchthat the second cycle generates a phosphorus linkage of a differentstereochemical configuration. In some embodiments, the stereochemistryof the linkage phosphorus in the newly formed internucleotidic linkageis controlled by using stereochemically pure phosphoramidites.Additionally, the modification reagent used in the modifying step of asubsequent cycle can be different from the modification reagent used inthe first or former cycle. The cumulative effect of this iterativeassembly approach is such that each component of a providedoligonucleotide can be structurally and configurationally tailored to ahigh degree. An additional advantage to this approach is that the stepof capping minimizes the formation of “n-1” impurities that wouldotherwise make isolation of a provided oligonucleotide extremelychallenging, and especially oligonucleotides of longer lengths.

In some embodiments, an exemplary cycle of the method for makingchirally controlled oligonucleotides is illustrated in Scheme I. InScheme I,

represents the solid support, and optionally a portion of the growingchirally controlled oligonucleotide attached to the solid support. Thechiral auxiliary exemplified has the structure of formula 3-I:

which is further described below. “Cap” is any chemical moietyintroduced to the nitrogen atom by the capping step, and in someembodiments, is an amino protecting group. One of ordinary skill in theart understands that in the first cycle, there may be only onenucleoside attached to the solid support when started, and cycle exitcan be performed optionally before deblocking. As understood by a personof skill in the art, B^(PRO) is a protected base used in oligonucleotidesynthesis. Each step of the above-depicted cycle of Scheme I isdescribed further below.

Synthesis on Solid Support

In some embodiments, the synthesis of a provided oligonucleotide isperformed on solid phase. In some embodiments, reactive groups presenton a solid support are protected. In some embodiments, reactive groupspresent on a solid support are unprotected. During oligonucleotidesynthesis a solid support is treated with various reagents in severalsynthesis cycles to achieve the stepwise elongation of a growingoligonucleotide chain with individual nucleotide units. The nucleosideunit at the end of the chain which is directly linked to the solidsupport is termed “the first nucleoside” as used herein. A firstnucleoside is bound to a solid support via a linker moiety, i.e. adiradical with covalent bonds between either of a CPG, a polymer orother solid support and a nucleoside. The linker stays intact during thesynthesis cycles performed to assemble the oligonucleotide chain and iscleaved after the chain assembly to liberate the oligonucleotide fromthe support.

Solid supports for solid-phase nucleic acid synthesis include thesupports described in, e.g., U.S. Pat. Nos. 4,659,774 , 5,141,813,4,458,066; Caruthers U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707,4,668,777, 4,973,679, and 5,132,418; Andrus et al. U.S. Pat. Nos.5,047,524, 5,262,530; and Koster U.S. Pat. Nos. 4,725,677 (reissued asRe34,069). In some embodiments, a solid phase is an organic polymersupport. In some embodiments, a solid phase is an inorganic polymersupport. In some embodiments, an organic polymer support is polystyrene,aminomethyl polystyrene, a polyethylene glycol-polystyrene graftcopolymer, polyacrylamide, polymethacrylate, polyvinylalcohol, highlycross-linked polymer (HCP), or other synthetic polymers, carbohydratessuch as cellulose and starch or other polymeric carbohydrates, or otherorganic polymers and any copolymers, composite materials or combinationof the above inorganic or organic materials. In some embodiments, aninorganic polymer support is silica, alumina, controlled polyglass(CPG), which is a silica-gel support, or aminopropyl CPG. Other usefulsolid supports include fluorous solid supports (see e.g.,WO/2005/070859), long chain alkylamine (LCAA) controlled pore glass(CPG) solid supports (see e.g., S. P. Adams, K. S. Kavka, E. J. Wykes,S. B. Holder and G. R. Galluppi, J. Am. Chem. Soc., 1983, 105, 661-663;G. R. Gough, M. J. Bruden and P. T. Gilham, Tetrahedron Lett., 1981, 22,4177-4180). Membrane supports and polymeric membranes (see e.g.Innovation and Perspectives in Solid Phase Synthesis, Peptides, Proteinsand Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton and U.S. Pat.No. 4,923,901) are also useful for the synthesis of nucleic acids. Onceformed, a membrane can be chemically functionalized for use in nucleicacid synthesis. In addition to the attachment of a functional group tothe membrane, the use of a linker or spacer group attached to themembrane is also used in some embodiments to minimize steric hindrancebetween the membrane and the synthesized chain.

Other suitable solid supports include those generally known in the artto be suitable for use in solid phase methodologies, including, forexample, glass sold as PrimerTM 200 support, controlled pore glass(CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., NucleicAcids Research, 1991, 19, 1527), TentaGel Support-anaminopolyethyleneglycol derivatized support (see, e.g., Wright, et al.,Tetrahedron Lett., 1993, 34, 3373), and Poros-a copolymer ofpolystyrene/divinylbenzene.

Surface activated polymers have been demonstrated for use in synthesisof natural and modified nucleic acids and proteins on several solidsupports mediums. A solid support material can be any polymer suitablyuniform in porosity, having sufficient amine content, and sufficientflexibility to undergo any attendant manipulations without losingintegrity. Examples of suitable selected materials include nylon,polypropylene, polyester, polytetrafluoroethylene, polystyrene,polycarbonate, and nitrocellulose. Other materials can serve as a solidsupport, depending on the design of the investigator. In considerationof some designs, for example, a coated metal, in particular gold orplatinum can be selected (see e.g., U.S. publication No. 20010055761).In one embodiment of oligonucleotide synthesis, for example, anucleoside is anchored to a solid support which is functionalized withhydroxyl or amino residues. Alternatively, a solid support isderivatized to provide an acid labile trialkoxytrityl group, such as atrimethoxytrityl group (TMT). Without being bound by theory, it isexpected that the presence of a trialkoxytrityl protecting group willpermit initial detritylation under conditions commonly used on DNAsynthesizers. For a faster release of oligonucleotide material insolution with aqueous ammonia, a diglycoate linker is optionallyintroduced onto the support.

In some embodiments, a provided oligonucleotide alternatively issynthesized from the 5′ to 3′ direction. In some embodiments, a nucleicacid is attached to a solid support through its 5′ end of the growingnucleic acid, thereby presenting its 3′ group for reaction, i.e. using5′-nucleoside phosphoramidites or in enzymatic reaction (e.g. ligationand polymerization using nucleoside 5′-triphosphates). When consideringthe 5′ to 3′ synthesis the iterative steps of the present inventionremain unchanged (i.e. capping and modification on the chiralphosphorus).

Linking Moiety

A linking moiety or linker is optionally used to connect a solid supportto a compound comprising a free nucleophilic moiety. Suitable linkersare known such as short molecules which serve to connect a solid supportto functional groups (e.g., hydroxyl groups) of initial nucleosidesmolecules in solid phase synthetic techniques. In some embodiments, thelinking moiety is a succinamic acid linker, or a succinate linker(—CO—CH₂—CH₂—CO—), or an oxalyl linker (—CO—CO—). In some embodiments,the linking moiety and the nucleoside are bonded together through anester bond. In some embodiments, a linking moiety and a nucleoside arebonded together through an amide bond. In some embodiments, a linkingmoiety connects a nucleoside to another nucleotide or nucleic acid.Suitable linkers are disclosed in, for example, Oligonucleotides AndAnalogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991,Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon,R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28.

A linker moiety is used to connect a compound comprising a freenucleophilic moiety to another nucleoside, nucleotide, or nucleic acid.In some embodiments, a linking moiety is a phosphodiester linkage. Insome embodiments, a linking moiety is an H-phosphonate moiety. In someembodiments, a linking moiety is a modified phosphorus linkage asdescribed herein. In some embodiments, a universal linker (UnyLinker) isused to attached the oligonucleotide to the solid support (Ravikumar etal., Org. Process Res. Dev., 2008, 12 (3), 399-410). In someembodiments, other universal linkers are used (Pon, R. T., Curr. Prot.Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, variousorthogonal linkers (such as disulfide linkers) are used (Pon, R. T.,Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

General Conditions—Solvents for Synthesis

Syntheses of provided oligonucleotides are generally performed inaprotic organic solvents. In some embodiments, a solvent is a nitrilesolvent such as, e.g., acetonitrile. In some embodiments, a solvent is abasic amine solvent such as, e.g., pyridine. In some embodiments, asolvent is an ethereal solvent such as, e.g., tetrahydrofuran. In someembodiments, a solvent is a halogenated hydrocarbon such as, e.g.,dichloromethane. In some embodiments, a mixture of solvents is used. Incertain embodiments a solvent is a mixture of any one or more of theabove-described classes of solvents.

In some embodiments, when an aprotic organic solvent is not basic, abase is present in the reacting step. In some embodiments where a baseis present, the base is an amine base such as, e.g., pyridine,quinoline, or N,N-dimethylaniline. Exemplary other amine bases includepyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline,N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

In some embodiments, a base is other than an amine base.

In some embodiments, an aprotic organic solvent is anhydrous. In someembodiments, an anhydrous aprotic organic solvent is freshly distilled.In some embodiments, a freshly distilled anhydrous aprotic organicsolvent is a basic amine solvent such as, e.g., pyridine. In someembodiments, a freshly distilled anhydrous aprotic organic solvent is anethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, afreshly distilled anhydrous aprotic organic solvent is a nitrile solventsuch as, e.g., acetonitrile.

Activation

An achiral H-phosphonate moiety is treated with the first activatingreagent to form the first intermediate. In one embodiment, the firstactivating reagent is added to the reaction mixture during thecondensation step. Use of the first activating reagent is dependent onreaction conditions such as solvents that are used for the reaction.Examples of the first activating reagent are phosgene, trichloromethylchloroformate, bis(trichloromethyl)carbonate (BTC), oxalyl chloride,Ph₃PCl₂, (PhO)₃PCl₂, N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BopCl),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP).

The example of achiral H-phosphonate moiety is a compound shown in theabove Scheme. DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene. H⁺DBUmay be, for example, ammonium ion, alkylammonium ion, heteroaromaticiminium ion, or heterocyclic iminium ion, any of which is primary,secondary, tertiary or quaternary, or a monovalent metal ion.

Reacting with Chiral Reagent

After the first activation step, the activated achiral H-phosphonatemoiety reacts with a chiral reagent, which is represented by formula(Z—I) or (Z—I′), to form a chiral intermediate of formula (Z—Va),(Z—Vb), (Z—Va′), or (Z—Vb′).

Stereospecific Condensation Step

A chiral intermediate of Formula Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) istreated with the second activating reagent and a nucleoside to form acondensed intermediate. The nucleoside may be on solid support. Examplesof the second activating reagent are 4,5-dicyanoimidazole (DCI),4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole(NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole(BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate(CMPT), N-cyanomethylpiperidinium triflate,N-cyanomethyldimethylammonium triflate. A chiral intermediate of FormulaZ—Va ((Z—Vb), (Z—Va′), or (Z—Vb′)) may be isolated as a monomer.Usually, the chiral intermediate of Z—Va ((Z—Vb), (Z—Va′), or (Z—Vb′))is not isolated and undergoes a reaction in the same pot with anucleoside or modified nucleoside to provide a chiral phosphitecompound, a condensed intermediate. In other embodiments, when themethod is performed via solid phase synthesis, the solid supportcomprising the compound is filtered away from side products, impurities,and/or reagents.

Capping Step

If the final nucleic acid is larger than a dimer, the unreacted —OHmoiety is capped with a blocking group and the chiral auxiliary in thecompound may also be capped with a blocking group to form a cappedcondensed intermediate. If the final nucleic acid is a dimer, then thecapping step is not necessary.

Modifying Step

The compound is modified by reaction with an electrophile. The cappedcondensed intermediate may be executed modifying step. In someembodiments, the modifying step is performed using a sulfurelectrophile, a selenium electrophile or a boronating agent. Examples ofmodifying steps are step of oxidation and sulfurization.

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas:S₈(Formula Z-B), Z^(z1)—S—S—Z^(z2), or Z^(z1)—S—V^(z)—Z^(z2);wherein Z^(zl) and Z^(z2) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z1) and Z^(z2) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, O, or NR^(f); and R^(f) is hydrogen, alkyl,alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof following Formulae Z—A, Z—B, Z—C, Z—D, Z—E, or Z—F:

In some embodiments, the selenium electrophile is a compound having oneof the following formulae:Se (Formula Z—G), Z^(z3)—Se—Se—Z^(z4), or Z^(z3)—Se—V^(z)—Z^(z4);wherein Z^(z3) and Z^(z4) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z3) and Z^(z4) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, S, O, or NR^(f); and R^(f) is hydrogen,alkyl, alkenyl, alkynyl, or aryl.

In some embodiments, the selenium electrophile is a compound of FormulaZ—G, Z—H, Z—I, Z—J, Z—K, or Z—L.

In some embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃ DIPEA), borane-pyridine (BH₃ Py),borane-2-chloropyridine (BH₃ CPy), borane-aniline (BH₃ An),borane-tetrahydrofiirane (BH₃ THF), or borane-dimethylsulfide (BH₃Me₂S).

In some embodiments of the method, the modifying step is an oxidationstep. In some embodiments of the method, the modifying step is anoxidation step using similar conditions as described above in thisapplication. In some embodiments, an oxidation step is as disclosed in,e.g., JP 2010-265304 A and WO2010/064146.

Chain Elongation Cycle and De-protection Step

The capped condensed intermediate is deblocked to remove the blockinggroup at the 5′-end of the growing nucleic acid chain to provide acompound. The compound is optionally allowed to re-enter the chainelongation cycle to form a condensed intermediate, a capped condensedintermediate, a modified capped condensed intermediate, and a5′-deprotected modified capped intermediate. Following at least oneround of chain elongation cycle, the 5′-deprotected modified cappedintermediate is further deblocked by removal of the chiral auxiliaryligand and other protecting groups for, e.g., nucleobase, modifiednucleobase, sugar and modified sugar protecting groups, to provide anucleic acid. In other embodiments, the nucleoside comprising a 5′-OHmoiety is an intermediate from a previous chain elongation cycle asdescribed herein. In yet other embodiments, the nucleoside comprising a5′-OH moiety is an intermediate obtained from another known nucleic acidsynthetic method. In embodiments where a solid support is used, thephosphorus-atom modified nucleic acid is then cleaved from the solidsupport. In certain embodiments, the nucleic acids is left attached onthe solid support for purification purposes and then cleaved from thesolid support following purification.

In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic method.In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic methodas described in this application. In yet other embodiments, thenucleoside comprising a 5′-OH moiety is an intermediate obtained fromanother known nucleic acid synthetic method comprising one or morecycles illustrated in Scheme I. In yet other embodiments, the nucleosidecomprising a 5′-OH moiety is an intermediate obtained from another knownnucleic acid synthetic method comprising one or more cycles illustratedin Scheme I-b, I-c or I-d.

In some embodiments, the present invention provides oligonucleotidesynthesis methods that use stable and commercially available materialsas starting materials. In some embodiments, the present inventionprovides oligonucleotide synthesis methods to produce stereocontrolledphosphorus atom-modified oligonucleotide derivatives using an achiralstarting material.

In some embodiments, the method of the present invention does not causedegradations under the de-protection steps. Further the method does notrequire special capping agents to produce phosphorus atom-modifiedoligonucleotide derivatives.

Condensing Reagent

Condensing reagents (C_(R)) useful in accordance with methods of thepresent invention are of any one of the following general formulae:

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, Z⁸, and Z⁹ are independentlyoptionally substituted group selected from alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, or heteroaryloxy, or wherein any of Z²and Z³, Z⁵ and Z⁶, Z⁷ and Z⁸, Z⁸ and Z⁹, Z⁹ and Z⁷, or Z⁷ and Z⁸ and Z⁹are taken together to form a 3 to 20 membered alicyclic or heterocyclicring; Q⁻ is a counter anion; and LG is a leaving group.

In some embodiments, a counter ion of a condensing reagent C_(R) is Cl⁻,Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, CIO₄ ⁻, or SbF₆ ⁻, wherein Tf isCF₃SO₂. In some embodiments, a leaving group of a condensing reagentC_(R) is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

Examples of condensing reagents used in accordance with methods of thepresent invention include, but are not limited to, pentafluorobenzoylchloride, carbonyldiimidazole (CDI),1-mesitylenesulfonyl-3-nitrotriazole (MSNT),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDCI—HCl),benzotriazole-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate(PyBOP), N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), and O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), DIPCDI;N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic bromide (BopBr),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP),3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP), bromotripyrrolidinophosphoniumhexafluorophosphate (PyBrOP);O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU); and tetramethylfluoroformamidinium hexafluorophosphate (TFFH).In certain embodiments, a counter ion of the condensing reagent C_(R) isCl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, whereinTf is CF₃SO₂.

In some embodiments, a condensing reagent is1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl) tetrazolide,pivaloyl chloride, bromotrispyrrolidinophosphonium hexafluorophosphate,N,N′-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl), or2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane. In some embodiment,a condensing reagent is N,N′-bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BopCl). In some embodiments, a condensing reagent is selectedfrom those described in WO/2006/066260).

In some embodiments, a condensing reagent is1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP):

Selection of Base and Sugar of Nucleoside Coupling Partner

As described herein, nucleoside coupling partners for use in accordancewith methods of the present invention can be the same as one another orcan be different from one another. In some embodiments, nucleosidecoupling partners for use in the synthesis of a provided oligonucleotideare of the same structure and/or stereochemical configuration as oneanother. In some embodiments, each nucleoside coupling partner for usein the synthesis of a provided oligonucleotide is not of the samestructure and/or stereochemical configuration as certain othernucleoside coupling partners of the oligonucleotide. Exemplarynucleobases and sugars for use in accordance with methods of the presentinvention are described herein. One of skill in the relevant chemicaland synthetic arts will recognize that any combination of nucleobasesand sugars described herein are contemplated for use in accordance withmethods of the present invention.

Coupling Step

Exemplary coupling procedures and chiral reagents and condensingreagents for use in accordance with the present invention are outlinedin, inter alia, Wada I (JP4348077; WO2005/014609; WO2005/092909), WadaII (WO2010/064146), and Wada III (WO2012/039448). Chiral nucleosidecoupling partners for use in accordance with the present invention arealso referred to herein as “Wada amidites.” In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase. In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase. Exemplary chiralphosphoramidites as coupling partner are depicted below:

One of the methods used for synthesizing the coupling partner isdepicted in Scheme II, below.

In some embodiments, the step of coupling comprises reacting a freehydroxyl group of a nucleotide unit of an oligonucleotide with anucleoside coupling partner under suitable conditions to effect thecoupling. In some embodiments, the step of coupling is preceded by astep of deblocking. For instance, in some embodiments, the 5′ hydroxylgroup of the growing oligonucleotide is blocked (i.e., protected) andmust be deblocked in order to subsequently react with a nucleosidecoupling partner.

Once the appropriate hydroxyl group of the growing oligonucleotide hasbeen deblocked, the support is washed and dried in preparation fordelivery of a solution comprising a chiral reagent and a solutioncomprising an activator. In some embodiments, a chiral reagent and anactivator are delivered simultaneously. In some embodiments, co-deliverycomprises delivering an amount of a chiral reagent in solution (e.g., aphosphoramidite solution) and an amount of activator in a solution(e.g., a CMPT solution) in a polar aprotic solvent such as a nitrilesolvent (e.g., acetonitrile).

In some embodiments, the step of coupling provides a crude productcomposition in which the chiral phosphite product is present in adiastereomeric excess of >95%. In some embodiments, the chiral phosphiteproduct is present in a diastereomeric excess of >96%. In someembodiments, the chiral phosphite product is present in a diastereomericexcess of >97%. In some embodiments, the chiral phosphite product ispresent in a diastereomeric excess of >98%. In some embodiments, thechiral phosphite product is present in a diastereomeric excess of >99%.

Capping Step

Provided methods for making chirally controlled oligonucleotidescomprise a step of capping. In some embodiments, a step of capping is asingle step. In some embodiments, a step of capping is two steps. Insome embodiments, a step of capping is more than two steps.

In some embodiments, a step of capping comprises steps of capping thefree amine of the chiral auxiliary and capping any residual unreacted 5′hydroxyl groups. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with the samecapping group. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with differentcapping groups. In certain embodiments, capping with different cappinggroups allows for selective removal of one capping group over the otherduring synthesis of the oligonucleotide. In some embodiments, thecapping of both groups occurs simultaneously. In some embodiments, thecapping of both groups occurs iteratively.

In certain embodiments, capping occurs iteratively and comprises a firststep of capping the free amine followed by a second step of capping thefree 5′ hydroxyl group, wherein both the free amine and the 5′ hydroxylgroup are capped with the same capping group. For instance, in someembodiments, the free amine of the chiral auxiliary is capped using ananhydride (e.g., phenoxyacetic anhydride, i.e., Pac₂O) prior to cappingof the 5′ hydroxyl group with the same anhydride. In certainembodiments, the capping of the 5′ hydroxyl group with the sameanhydride occurs under different conditions (e.g., in the presence ofone or more additional reagents). In some embodiments, capping of the 5′hydroxyl group occurs in the presence of an amine base in an etherialsolvent (e.g., NMI (N-methylimidazole) in THF). The phrase “cappinggroup” is used interchangeably herein with the phrases “protectinggroup” and “blocking group”.

In some embodiments, an amine capping group is characterized in that iteffectively caps the amine such that it prevents rearrangement and/ordecomposition of the intermediate phosphite species. In someembodiments, a capping group is selected for its ability to protect theamine of the chiral auxiliary in order to prevent intramolecularcleavage of the internucleotide linkage phosphorus.

In some embodiments, a 5′ hydroxyl group capping group is characterizedin that it effectively caps the hydroxyl group such that it prevents theoccurrence of “shortmers,” e.g., “n-m” (m and n are integers and m<n; nis the number of bases in the targeted oligonucleotide) impurities thatoccur from the reaction of an oligonucleotide chain that fails to reactin a first cycle but then reacts in one or more subsequent cycles. Thepresence of such shortmers, especially “n-1”, has a deleterious effectupon the purity of the crude oligonucleotide and makes finalpurification of the oligonucleotide tedious and generally low-yielding.

In some embodiments, a particular cap is selected based on its tendencyto facilitate a particular type of reaction under particular conditions.For instance, in some embodiments, a capping group is selected for itsability to facilitate an E1 elimination reaction, which reaction cleavesthe cap and/or auxiliary from the growing oligonucleotide. In someembodiments, a capping group is selected for its ability to facilitatean E2 elimination reaction, which reaction cleaves the cap and/orauxiliary from the growing oligonucleotide. In some embodiments, acapping group is selected for its ability to facilitate a (β-eliminationreaction, which reaction cleaves the cap and/or auxiliary from thegrowing oligonucleotide.

Modifying Step

As used herein, the phrase “modifying step”, “modification step” and“P-modification step” are used interchangeably and refer generally toany one or more steps used to install a modified internucleotidiclinkage. In some embodiments, the modified internucleotidic linkagehaving the structure of formula I. A P-modification step of the presentinvention occurs during assembly of a provided oligonucleotide ratherthan after assembly of a provided oligonucleotide is complete. Thus,each nucleotide unit of a provided oligonucleotide can be individuallymodified at the linkage phosphorus during the cycle within which thenucleotide unit is installed.

In some embodiments, a suitable P-modification reagent is a sulfurelectrophile, selenium electrophile, oxygen electrophile, boronatingreagent, or an azide reagent.

For instance, in some embodiments, a selemium reagent is elementalselenium, a selenium salt, or a substituted diselenide. In someembodiments, an oxygen electrophile is elemental oxygen, peroxide, or asubstituted peroxide. In some embodiments, a boronating reagent is aborane-amine (e.g., N,N-diisopropylethylamine (BH₃·DIPEA),borane-pyridine (BH₃·Py), borane-2-chloropyridine (BH₃·CPy),borane-aniline (BH₃·An)), a borane-ether reagent (e.g.,borane-tetrahydrofuran (BH₃·THF)), a borane-dialkylsulfide reagent(e.g., BH₃·Me₂S), aniline-cyanoborane, or atriphenylphosphine-carboalkoxyborane. In some embodiments, an azidereagent is comprises an azide group capable of undergoing subsequentreduction to provide an amine group.

In some embodiments, a P-modification reagent is a sulfurization reagentas described herein. In some embodiments, a step of modifying comprisessulfurization of phosphorus to provide a phosphorothioate linkage orphosphorothioate triester linkage. In some embodiments, a step ofmodifying provides an oligonucleotide having an internucleotidic linkageof formula I.

In some embodiments, the present invention provides sulfurizingreagents, and methods of making, and use of the same.

In some embodiments, such sulfurizing reagents are thiosulfonatereagents. In some embodiments, a thiosulfonate reagent has a structureof formula S-I:

wherein:R^(s1) is R; andeach of R, L and R¹ is independently as defined and described above andherein.

In some embodiments, the sulfurizing reagent is a bis(thiosulfonate)reagent. In some embodiments, the bis(thiosulfonate) reagent has thestructure of formula S-II:

wherein each of R^(s1) and L is independently as defined and describedabove and herein.

As defined generally above, R^(s1) is R, wherein R is as defined anddescribed above and herein. In some embodiments, R^(s1) is optionallysubstituted aliphatic, aryl, heterocyclyl or heteroaryl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is methyl. In some embodiments, R^(s1) iscyanomethyl. In some embodiments, R^(s1) is nitromethyl. In someembodiments, R^(s1) is optionally substituted aryl. In some embodiments,R^(s1) is optionally substituted phenyl. In some embodiments, R^(s1) isphenyl. In some embodiments, R^(s1) is p-nitrophenyl. In someembodiments, R^(s1) is p-methylphenyl. In some embodiments, R^(s1) isp-chlorophenyl. In some embodiments, R^(s1) is o-chlorophenyl. In someembodiments, R^(s1) is 2,4,6-trichlorophenyl. In some embodiments,R^(s1) is pentafluorophenyl. In some embodiments, R^(s1) is optionallysubstituted heterocyclyl. In some embodiments, R^(s1) is optionallysubstituted heteroaryl.

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S—is

In some embodiments, R¹—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is —S—R^(L3)— or —S—C(O)—R^(L3)—. In some embodiments, Lis —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkenylene. In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—,wherein R^(L3) is an optionally substituted C₁-C₆ alkylene wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkenylene, arylene, or heteroarylene. Insome embodiments, In some embodiments, R^(L3) is an optionallysubstituted —S—(C₁-C₆ alkenylene), —S—(C₁-C₆ alkylene)—, —S—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene), —S—CO—arylene-(C₁-C₆ alkylene)—, or—S—CO—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)—. In some embodiments,the sulfurizing reagent has the structure of S-I or S-II, wherein L is—S—R^(L3)— or —S—C(O)—R^(L3)—, and the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I or—S-II, wherein L is alkylene, alkenylene, arylene or heteroarylene.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

In some embodiments, L is

wherein the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is

In some embodiments, R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

wherein the sulfur atom is connected to R¹; and R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is —S—R^(L2), wherein R^(L2) is as defined anddescribed above and herein. In some embodiments, R^(L2) is an optionallysubstituted group selected from —S—(C₁-C₆ alkylene)-heterocyclyl,—S—(C₁-C₆ alkenylene)-heterocyclyl, —S—(C₁-C₆ alkylene—N(R′)₂, —S—(C₁-C₆alkylene)—N(R′)₃, wherein each R′ is as defined above and describedherein.

In some embodiments, —L—R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, —L—R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

Exemplary bis(thiosulfonate) reagents of formula S-II are depictedbelow:

In some embodiments, the sulfurization reagent is a compound having oneof the following formulae:S_(g), R^(s2)—S—S—R^(s3), or R^(s2)—S—X^(s)—R^(s3),wherein:each of R^(s2) and R^(s3) is independently an optionally substitutedgroup selected from aliphatic, aminoalkyl, carbocyclyl, heterocyclyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy,acyl, amide, imide, or thiocarbonyl; or

R^(s2) and R^(s3) are taken together with the atoms to which they arebound to form an optionally substituted heterocyclic or heteroaryl ring;

X^(s) is —S(O)₂—, —O—, or —N(R′); and

R′ is as defined and described above and herein.

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is

Exemplary sulfuring reagents are depicted in Table 5 below.

TABLE 5 Exemplary sulfurization reagents.

S₈

In some embodiments, a provided sulfurization reagent is used to modifyan H-phosphonate. For instance, in some embodiments, an H-phosphonateoligonucleotide is synthesized using, e.g., a method of Wada I or WadaII, and is modified using a sulfurization reagent of formula S-I orS-II:

wherein each of R^(S1), L, and R¹ are as described and defined above andherein.

In some embodiments, the present invention provides a process forsynthesizing a phosphorothioate triester, comprising steps of:

-   -   i) reacting an H-phosphonate of structure:

-   -   wherein each of W, Y, and Z are as described and defined above        and herein, with a silylating reagent to provide a        silyloxyphosphonate; and    -   ii) reacting the silyloxyphosphonate with a sulfurization        reagent of structure S-I or S-II:

-   -   to provide a phosphorothiotriester.

In some embodiments, a selenium electrophile is used instead of asulfurizing reagent to introduce modification to the internucleotidiclinkage. In some embodiments, a selenium electrophile is a compoundhaving one of the following formulae:Se, R^(s2)—Se—Se—R^(s3), or R^(s2)—Se—X^(s)—R^(s3),wherein:each of R^(s2) and R^(s3) is independently an optionally substitutedgroup selected from aliphatic, aminoalkyl, carbocyclyl, heterocyclyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy,acyl, amide, imide, or thiocarbonyl; or

R^(s2) and R^(s3) are taken together with the atoms to which they arebound to form an optionally substituted heterocyclic or heteroaryl ring;

X^(s) is —S(O)₂—, —O—, or —N(R′)—; and

R′ is as defined and described above and herein.

In other embodiments, the selenium electrophile is a compound of Se,KSeCN,

In some embodiments, the selenium electrophile is Se or

In some embodiments, a sulfurization reagent for use in accordance withthe present invention is characterized in that the moiety transferred tophosphorus during sulfurization is a substituted sulfur (e.g., —SR) asopposed to a single sulfur atom (e.g., —S⁻ ═S).

In some embodiments, a sulfurization reagent for use in accordance withthe present invention is characterized in that the activity of thereagent is tunable by modifying the reagent with a certain electronwithdrawing or donating group.

In some embodiments, a sulfurization reagent for use in accordance withthe present invention is characterized in that it is crystalline. Insome embodiments, a sulfurization reagent for use in accordance with thepresent invention is characterized in that it has a high degree ofcrystallinity. In certain embodiments, a sulfurization reagent for usein accordance with the present invention is characterized by ease ofpurification of the reagent via, e.g., recrystallization. In certainembodiments, a sulfurization reagent for use in accordance with thepresent invention is characterized in that it is substantially free fromsulfur-containing impurities. In some embodiments, sulfurizationreagents which are substantially free from sulfur-containing impuritiesshow increased efficiency.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages. To synthesize suchchirally controlled oligonucleotides, one or more modifying steps areoptionally replaced with an oxidation step to install the correspondingphosphate diester linkages. In some embodiments, the oxidation step isperformed in a fashion similar to ordinary oligonucleotide synthesis. Insome embodiments, an oxidation step comprises the use of I₂. In someembodiments, an oxidation step comprises the use of I₂ and pyridine. Insome embodiments, an oxidation step comprises the use of 0.02 M I₂ in aTHF/pyridine/water (70:20:10-v/v/v) co-solvent system. An exemplarycycle is depicted in Scheme I-c.

In some embodiments, a phosphorothioate precursor is used to synthesizechirally controlled oligonucleotides comprising phosphorothioatelinkages. In some embodiments, such a phosphorothioate precursor is

In some embodiments,

is converted into phosphorothioate diester linkages during standarddeprotection/release procedure after cycle exit. Examples are furtherdepicted below.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages and one or morephosphorothioate diester linkages. In some embodiments, the providedchirally controlled oligonucleotide comprises one or more phosphatediester linkages and one or more phosphorothioate diester linkages,wherein at least one phosphate diester linkage is installed after allthe phosphorothioate diester linkages when synthesized from 3′ to 5′. Tosynthesize such chirally controlled oligonucleotides, in someembodiments, one or more modifying steps are optionally replaced with anoxidation step to install the corresponding phosphate diester linkages,and a phosphorothioate precursor is installed for each of thephosphorothioate diester linkages. In some embodiments, aphosphorothioate precursor is converted to a phosphorothioate diesterlinkage after the desired oligonucleotide length is achieved. In someembodiments, the deprotection/release step during or after cycle exitconverts the phosphorothioate precursors into phosphorothioate diesterlinkages. In some embodiments, a phosphorothioate precursor ischaracterized in that it has the ability to be removed by abeta-elimination pathway. In some embodiments, a phosphorothioateprecursor is

As understood by one of ordinary skill in the art, one of the benefitsof using a phosphorothioate precursor, for instance,

during synthesis is that

is more stable than phosphorothioate in certain conditions.

In some embodiments, a phosphorothioate precursor is a phosphorusprotecting group as described herein, e.g., 2-cyanoethyl (CE or Cne),2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl,o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl,3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl,2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl,2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl. Examples are furtherdepicted below.

Methods for synthesizing a desired sulfurization reagent are describedherein and in the examples section.

As noted above, in some embodiments, sulfurization occurs underconditions which cleave the chiral reagent from the growingoligonucleotide. In some embodiments, sulfurization occurs underconditions which do not cleave the chiral reagent from the growingoligonucleotide.

In some embodiments, a sulfurization reagent is dissolved in a suitablesolvent and delivered to the column. In certain embodiments, the solventis a polar aprotic solvent such as a nitrile solvent. In someembodiments, the solvent is acetonitrile. In some embodiments, asolution of sulfurization reagent is prepared by mixing a sulfurizationreagent (e.g., a thiosulfonate derivative as described herein) withBSTFA (N,O-bis-trimethylsilyl-trifluoroacetamide) in a nitrile solvent(e.g., acetonitrile). In some embodiments, BSTFA is not included. Forexample, the present inventors have found that relatively more reactivesulfurization reagents of general formula R^(s2)—S—S(O)₂—R^(s3) canoften successfully participate in sulfurization reactions in the absenceof BSTFA. To give but one example, the inventors have demonstrated thatwhere R^(s2) is p-nitrophenyl and R^(s3) is methyl then no BSTFA isrequired. In light of this disclosure, those skilled in the art willreadily be able to determine other situations and/or sulfurizationreagents that do not require BSTFA.

In some embodiments, the sulfurization step is performed at roomtemperature. In some embodiments, the sulfurization step is performed atlower temperatures such as about 0° C., about 5° C., about 10° C., orabout 15° C. In some embodiments, the sulfurization step is performed atelevated temperatures of greater than about 20° C.

In some embodiments, a sulfurization reaction is run for about 1 minuteto about 120 minutes. In some embodiments, a sulfurization reaction isrun for about 1 minute to about 90 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 60 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 30 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 25 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 20 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 10 minutes. In some embodiments, asulfurization reaction is run for about 5 minute to about 60 minutes.

In some embodiments, a sulfurization reaction is run for about 5minutes. In some embodiments, a sulfurization reaction is run for about10 minutes. In some embodiments, a sulfurization reaction is run forabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 20 minutes. In some embodiments, a sulfurization reaction isrun for about 25 minutes. In some embodiments, a sulfurization reactionis run for about 30 minutes. In some embodiments, a sulfurizationreaction is run for about 35 minutes. In some embodiments, asulfurization reaction is run for about 40 minutes. In some embodiments,a sulfurization reaction is run for about 45 minutes. In someembodiments, a sulfurization reaction is run for about 50 minutes. Insome embodiments, a sulfurization reaction is run for about 55 minutes.In some embodiments, a sulfurization reaction is run for about 60minutes.

It was unexpectedly found that certain of the sulfurization modificationproducts made in accordance with methods of the present invention areunexpectedly stable. In some embodiments, it the unexpectedly stableproducts are phosphorothioate triesters. In some embodiments, theunexpectedly stable products are chirally controlled oligonucleotidescomprising one or more internucleotidic linkages having the structure offormula I-c.

One of skill in the relevant arts will recognize that sulfurizationmethods described herein and sulfurization reagents described herein arealso useful in the context of modifying H-phosphonate oligonucleotidessuch as those described in Wada II (WO2010/064146).

In some embodiments, the sulfurization reaction has a stepwisesulfurization efficiency that is at least about 80%, 85%, 90%, 95%, 96%,97%, or 98%. In some embodiments, the sulfurization reaction provides acrude dinucleotide product compositon that is at least 98% pure. In someembodiments, the sulfurization reaction provides a crude tetranucleotideproduct compositon that is at least 90% pure. In some embodiments, thesulfurization reaction provides a crude dodecanucleotide productcompositon that is at least 70% pure. In some embodiments, thesulfurization reaction provides a crude icosanucleotide productcompositon that is at least 50% pure.

Once the step of modifying the linkage phosphorus is complete, theoligonucleotide undergoes another deblock step in preparation forre-entering the cycle. In some embodiments, a chiral auxiliary remainsintact after sulfurization and is deblocked during the subsequentdeblock step, which necessarily occurs prior to re-entering the cycle.The process of deblocking, coupling, capping, and modifying, arerepeated until the growing oligonucleotide reaches a desired length, atwhich point the oligonucleotide can either be immediately cleaved fromthe solid support or left attached to the support for purificationpurposes and later cleaved. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleaveageof the oligonucleotide from the support and deprotection of the basesoccurs in a single step. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleaveageof the oligonucleotide from the support and deprotection of the basesoccurs in more than one steps. In some embodiments, deprotection andcleavage from the support occurs under basic conditions using, e.g., oneor more amine bases. In certain embodiments, the one or more amine basescomprise propyl amine. In certain embodiments, the one or more aminebases comprise pyridine.

In some embodiments, cleavage from the support and/or deprotectionoccurs at elevated temperatures of about 30° C. to about 90° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 40° C. to about 80° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 50° C. to about 70° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 60° C. In some embodiments, cleavage fromthe support and/or deprotection occurs at ambient temperatures.

Exemplary purification procedures are described herein and/or are knowngenerally in the relevant arts.

Noteworthy is that the removal of the chiral auxiliary from the growingoligonucleotide during each cycle is beneficial for at least the reasonsthat (1) the auxiliary will not have to be removed in a separate step atthe end of the oligonucleotide synthesis when potentially sensitivefunctional groups are installed on phosphorus; and (2) unstablephosphorus- auxiliary intermediates prone to undergoing side reactionsand/or interfering with subsequent chemistry are avoided. Thus, removalof the chiral auxiliary during each cycle makes the overall synthesismore efficient.

While the step of deblocking in the context of the cycle is describedabove, additional general methods are included below.

Deblocking Step

In some embodiments, the step of coupling is preceded by a step ofdeblocking. For instance, in some embodiments, the 5′ hydroxyl group ofthe growing oligonucleotide is blocked (i.e., protected) and must bedeblocked in order to subsequently react with a nucleoside couplingpartner.

In some embodiments, acidification is used to remove a blocking group.In some embodiments, the acid is a Bronsted acid or Lewis acid. UsefulBronsted acids are carboxylic acids, alkylsulfonic acids, arylsulfonicacids, phosphoric acid and its derivatives, phosphonic acid and itsderivatives, alkylphosphonic acids and their derivatives, arylphosphonicacids and their derivatives, phosphinic acid, dialkylphosphinic acids,and diarylphosphinic acids which have a pKa (25° C. in water) value of−0.6 (trifluoroacetic acid) to 4.76 (acetic acid) in an organic solventor water (in the case of 80% acetic acid). The concentration of the acid(1 to 80%) used in the acidification step depends on the acidity of theacid. Consideration to the acid strength must be taken into account asstrong acid conditions will result in depurination/depyrimidination,wherein purinyl or pyrimidinyl bases are cleaved from ribose ring and orother sugar ring. In some embodiments, an acid is selected fromR^(a1)COOH, R^(a1)SO₃H, R^(a3)SO₃H,

wherein each of R^(a1) and R^(a2) is independently hydrogen or anoptionally substituted alkyl or aryl, and R^(a1) is an optionallysubstituted alkyl or aryl.

In some embodiments, acidification is accomplished by a Lewis acid in anorganic solvent. Exemplary such useful Lewis acids are Zn(X^(a))₂wherein X^(a) is Cl, Br, I, or CF₃SO₃.

In some embodiments, the step of acidifying comprises adding an amountof a Bronsted or Lewis acid effective to remove a blocking group withoutremoving purine moieties from the condensed intermediate.

Acids that are useful in the acidifying step also include, but are notlimited to 10% phosphoric acid in an organic solvent, 10% hydrochloricacid in an organic solvent, 1% trifluoroacetic acid in an organicsolvent, 3% dichloroacetic acid or trichloroacetic acid in an organicsolvent or 80% acetic acid in water. The concentration of any Bronstedor Lewis acid used in this step is selected such that the concentrationof the acid does not exceed a concentration that causes cleavage of anucleobase from a sugar moiety.

In some embodiments, acidification comprises adding 1% trifluoroaceticacid in an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 8% trifluoroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 3%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding about 0.1% to about 10%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding 3% trichloroacetic acidin an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 10% trichloroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 80% aceticacid in water. In some embodiments, acidification comprises adding about50% to about 90%, or about 50% to about 80%, about 50% to about 70%,about 50% to about 60%, about 70% to about 90% acetic acid in water. Insome embodiments, the acidification comprises the further addition ofcation scavengers to an acidic solvent. In certain embodiments, thecation scavengers can be triethylsilane or triisopropylsilane. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 1% trifluoroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% dichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in dichloromethane.

In certain embodiments, methods of the present invention are completedon a synthesizer and the step of deblocking the hydroxyl group of thegrowing oligonucleotide comprises delivering an amount solvent to thesynthesizer column, which column contains a solid support to which theoligonucleotide is attached. In some embodiments, the solvent is ahalogenated solvent (e.g., dichloromethane). In certain embodiments, thesolvent comprises an amount of an acid. In some embodiments, the solventcomprises an amount of an organic acid such as, for instance,trichloroacetic acid. In certain embodiments, the acid is present in anamount of about 1% to about 20% w/v. In certain embodiments, the acid ispresent in an amount of about 1% to about 10% w/v. In certainembodiments, the acid is present in an amount of about 1% to about 5%w/v. In certain embodiments, the acid is present in an amount of about 1to about 3% w/v. In certain embodiments, the acid is present in anamount of about 3% w/v. Methods for deblocking a hydroxyl group aredescribed further herein. In some embodiments, the acid is present in 3%w/v is dichloromethane.

In some embodiments, the chiral auxiliary is removed before thedeblocking step. In some embodiments, the chiral auxiliary is removedduring the deblocking step.

In some embodiments, cycle exit is performed before the deblocking step.In some embodiments, cycle exit is preformed after the deblocking step.

General Conditions for Blocking Group/Protecting Group Removal

Functional groups such as hydroxyl or amino moieties which are locatedon nucleobases or sugar moieties are routinely blocked with blocking(protecting) groups (moieties) during synthesis and subsequentlydeblocked. In general, a blocking group renders a chemical functionalityof a molecule inert to specific reaction conditions and can later beremoved from such functionality in a molecule without substantiallydamaging the remainder of the molecule (see e.g., Green and Wuts,Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, NewYork, 1991). For example, amino groups can be blocked with nitrogenblocking groups such as phthalimido, 9-fludrenylmethoxycarbonyl (FMOC),triphenylmethylsulfenyl, t-BOC, 4,4′-dimethoxytrityl (DMTr),4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or9-(p-methoxyphenyl)xanthin-9-yl (MOX). Carboxyl groups can be protectedas acetyl groups. Hydroxy groups can be protected such astetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS),1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp),1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp),1-(2-chloroethoxy)ethyl, 3 -methoxy-1,5 -dicarbomethoxypentan-3-yl(MDP), bis(2-acetoxyethoxy)methyl (ACE), triisopropylsilyloxymethyl(TOM), 1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM),[4-(N-dichloroacetyl-N-methylamino)benzyloxy]methyl, 2-cyanoethyl (CN),pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other representativehydroxyl blocking groups have been described (see e.g., Beaucage et al.,Tetrahedron, 1992, 46, 2223). In some embodiments, hydroxyl blockinggroups are acid-labile groups, such as the trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups canalso be blocked by including them in a precursor form. Thus an azidogroup can be considered a blocked form of an amine as the azido group iseasily converted to the amine. Further representative protecting groupsutilized in nucleic acid synthesis are known (see e.g. Agrawal et al.,Protocols for Oligonucleotide Conjugates, Eds., Humana Press, NewJersey, 1994, Vol. 26, pp. 1-72).

Various methods are known and used for removal of blocking groups fromnucleic acids. In some embodiments, all blocking groups are removed. Insome embodiments, a portion of blocking groups are removed. In someembodiments, reaction conditions can be adjusted to selectively removecertain blocking groups.

In some embodiments, nucleobase blocking groups, if present, arecleavable with an acidic reagent after the assembly of a providedoligonucleotide. In some embodiment, nucleobase blocking groups, ifpresent, are cleavable under neither acidic nor basic conditions, e.g.cleavable with fluoride salts or hydrofluoric acid complexes. In someembodiments, nucleobase blocking groups, if present, are cleavable inthe presence of base or a basic solvent after the assembly of a providedoligonucleotide. In certain embodiments, one or more of the nucleobaseblocking groups are characterized in that they are cleavable in thepresence of base or a basic solvent after the assembly of a providedoligonucleotide but are stable to the particular conditions of one ormore earlier deprotection steps occurring during the assembly of theprovided oligonucleotide.

In some embodiments, blocking groups for nucleobases are not required.In some embodiments, blocking groups for nucleobases are required. Insome embodiments, certain nucleobases require one or more blockinggroups while other nucleobases do not require one or more blockinggroups.

In some embodiments, the oligonucleotide is cleaved from the solidsupport after synthesis. In some embodiments, cleavage from the solidsupport comprises the use of propylamine. In some embodiments, cleavagefrom the solid support comprises the use of propylamine in pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in pyridine. In some embodiments, cleavage from thesolid support comprises the use of propylamine in anhydrous pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in anhydrous pyridine. In some embodiments, cleavagefrom the solid support comprises use of a polar aprotic solvent such asacetonitrile, NMP, DMSO, sulfone, and/or lutidine. In some embodiments,cleavage from the solid support comprises use of solvent, e.g., a polaraprotic solvent, and one or more primary amines (e.g., a C₁-C₁₀ amine),and/or one or more of methoxylamine, hydrazine, and pure anhydrousammonia.

In some embodiments, deprotection of oligonucleotide comprises the useof propylamine. In some embodiments, deprotection of oligonucleotidecomprises the use of propylamine in pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inpyridine. In some embodiments deprotection of oligonucleotide comprisesthe use of propylamine in anhydrous pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inanhydrous pyridine.

In some embodiments, the oligonucleotide is deprotected during cleavage.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about room temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at elevated temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at above about 30° C., 40°C., 50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about 30° C., 40° C.,50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 40-80° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 50-70° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 60° C.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for more than 0.1 hr, 1hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 0.1-5 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 3-10 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5-15 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10-20 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15-25 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 20-40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 2 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 18 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 24 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature formore than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30hrs, or 40 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed at roomtemperature for about 5-48 hrs. In some embodiments, cleavage ofoligonucleotide from solid support, or deprotection of oligonucleotide,is performed at room temperature for about 10-24hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature forabout 18 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed atelevated temperature for more than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs,15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. In some embodiments, cleavageof oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at elevated temperature for about 0.5-5hrs. In some embodiments, cleavage of oligonucleotide from solidsupport, or deprotection of oligonucleotide, is performed at about 60°C. for about 0.5-5 hrs. In some embodiments, cleavage of oligonucleotidefrom solid support, or deprotection of oligonucleotide, is performed atabout 60° C. for about 2 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide comprises the use of propylamine and isperformed at room temperature or elevated temperature for more than 0.1hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40hrs. Exemplary conditions are 20% propylamine in pyridine at roomtemperature for about 18 hrs, and 20% propylamine in pyridine at 60° C.for about 18 hrs,

In some embodiments, an activator is a “Wada”—O—activator, i.e., theactivator is from any one of Wada I, II, or III documents cited above.

Exemplary activating groups are depicted below:

In some embodiments, an activating reagent is selected from

An exemplary cycle is depicted in Scheme I-b.

An exemplary cycle is illustrated in Scheme I-c.

In Scheme I-c, oligonucleotide (or nucleotide, or oligonucleotide withmodified internucleotidic linkage) on solid support (C-1) is coupledwith phosphoramidite C-2. After coupling and capping, an oxidation stepis performed. After deblocking, a phosphate diester linkage is formed.The cycle product C-3 can either re-enter cycle C to install morephosphate diester linkage, or enter other cycles to install other typesof internucleotidic linkages, or go to cycle exit.

In some embodiments, non-chirally pure phosphoramidite can be usedinstead of C-2 in Scheme I-c. In some embodiments,β-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, the use of a phosphorothioate diester precursorincreases the stability of oligonucleotide during synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe efficiency of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe yield of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe product purity of chirally controlled oligonucleotide synthesis.

In some embodiments, the phosphorothioate diester precursor in theabove-mentioned methods is

In some embodiments,

is converted to a phosphorothioate diester linkage duringdeprotection/release. An exemplary cycle is depicted in Scheme I-d. Moreexamples are depicted below.

As illustrated in Scheme I-d, both phosphorothioate and phosphatediester linkages can be incorporated into the same chirally controlledoligonucleotide. As understood by a person of ordinary skill in the art,the provided methods do not require that the phosphorothioate diesterand the phosphate diester to be consecutive—other internucleotidiclinkages can form between them using a cycle as described above. InScheme I-d, phosphorothioate diester precursors,

are installed in place of the phosphorothioate diester linkages. In someembodiments, such replacement provided increased synthesis efficiencyduring certain steps, for instance, the oxidation step. In someembodiments, the use of phosphorothioate diester precursors generallyimprove the stability of chirally controlled oligonucleotides duringsynthesis and/or storage. After cycle exit, during deprotection/release,the phosphorothioate diester precursor is converted to phosphorothioatediester linkage. In some embodiments, it is benefical to usephosphorothioate diester precursor even when no phosphate diesterlinkage is present in the chirally controlled oligonucleotide, or nooxidation step is required during synthesis.

As in Scheme I-c, in some embodiments, non-chirally pure phosphoramiditecan be used for cycles comprising oxidation steps. In some embodiments,β-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the

phosphoramidite being used has the structure of

In some embodiments, methods of the present invention provide chirallycontrolled oligonucleotide compositions that are enriched in aparticular oligonucleotide type.

In some embodiments, at least about 10% of a provided crude compositionis of a particular oligonucleotide type. In some embodiments, at leastabout 20% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 30% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 50% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 60% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 70% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 80% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 90% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided crude composition isof a particular oligonucleotide type.

In some embodiments, at least about 1% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 2%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 3% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 4%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 5% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 10%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 20% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 30%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 50%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 60% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 70%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 80% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 90%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided composition is of aparticular oligonucleotide type.

Biological Applications and Exemplary Use

Among other things, the present invention recognizes that properties andactivities of an oligonucleotide can be adjusted by optimizing itspattern of backbone chiral centers through the use of provided chirallycontrolled oligonucleotide compositions. In some embodiments, thepresent invention provides chirally controlled oligonucleotidecompositions, wherein the oligonucleotides have a common pattern ofbackbone chiral centers which enhances their stability and/or biologicalactivity. In some embodiments, a pattern of backbone chiral centersprovides unexpectedly increased stability. In some embodiments, apattern of backbone chiral centers, surprisingly, provides greatlyincreased activity. In some embodiments, a pattern of backbone chiralcenters provides both increased stability and activity. In someembodiments, when an oligonucleotide is utilized to cleave a nucleicacid polymer, a pattern of backbone chiral centers of theoligonucleotide, surprisingly by itself, changes the cleavage pattern ofa target nucleic acid polymer. In some embodiments, a pattern ofbackbone chiral centers effectively prevents cleavage at secondarysites. In some embodiments, a pattern of backbone chiral centers createsnew cleavage sites. In some embodiments, a pattern of backbone chiralcenters minimizes the number of cleavage sites. In some embodiments, apattern of backbone chiral centers minimizes the number of cleavagesites so that a target nucleic acid polymer is cleaved at only one sitewithin the sequence of the target nucleic acid polymer that iscomplementary to the oligonucleotide. In some embodiments, a pattern ofbackbone chiral centers enhances cleavage efficiency at a cleavage site.In some embodiments, a pattern of backbone chiral centers of theoligonucleotide improves cleavage of a target nucleic acid polymer. Insome embodiments, a pattern of backbone chiral centers increasesselectivity. In some embodiments, a pattern of backbone chiral centersminimizes off-target effect. In some embodiments, a pattern of backbonechiral centers increase selectivity, e.g., cleavage selectivity amongtarget sequences differing by point mutations or single nucleotidepolymorphisms (SNPs). In some embodiments, a pattern of backbone chiralcenters increase selectivity, e.g., cleavage selectivity among targetsequences differing by only one point mutation or single nucleotidepolymorphism (SNP).

In some embodiments, the present invention provides a method forcontrolled cleavage of a nucleic acid polymer, comprising providing achirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers; and        wherein the nucleic acid polymer is cleaved in a cleavage        pattern that is different than the cleavage pattern when        chirally uncontrolled oligonucleotide composition is provided.

As used herein, a cleavage pattern of a nucleic acid polymer is definedby the number of cleavage sites, the locations of the cleavage sites,and the percentage of cleavage at each sites. In some embodiments, acleavage pattern has multiple cleavage sites, and the percentage ofcleavage at each site is different. In some embodiments, a cleavagepattern has multiple cleavage sites, and the percentage of cleavage ateach site is the same. In some embodiments, a cleavage pattern has onlyone cleavage site. In some embodiments, cleavage patterns differ fromeach other in that they have different numbers of cleavage sites. Insome embodiments, cleavage patterns differ from each other in that atleast one cleavage location is different. In some embodiments, cleavagepatterns differ from each other in that the percentage of cleavage at atleast one common cleavage site is different. In some embodiments,cleavage patterns differ from each other in that they have differentnumbers of cleavage sites, and/or at least one cleavage location isdifferent, and/or the percentage of cleavage at at least one commoncleavage site is different.

In some embodiments, the present invention provides a method forcontrolled cleavage of a nucleic acid polymer, the method comprisingsteps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type.

In some embodiments, the present invention provides a method forcontrolled cleavage of a nucleic acid polymer, the method comprisingsteps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type, the        contacting being performed under conditionsso that cleavage of        the nucleic acid polymer occurs.

In some embodiments, the present invention provides a method forchanging a first cleavage pattern of a nucleic acid polymer resultedfrom using a first oligonucleotide composition, comprising providing asecond chirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers; and        wherein the nucleic acid polymer is cleaved in a cleavage        pattern that is different than the first cleavage pattern.

In some embodiments, the present invention provides a method foraltering a cleavage pattern observed when a nucleic acid polymer whosenucleotide sequence includes a target sequence is contacted with areference oligonucleotide composition that comprises oligonucleotideshaving a particular base sequence and length, which particular basesequence is or comprises a sequence that is complementary to the targetsequence, the method comprising:

contacting the nucleic acid polymer with a chirally controlledoligonucleotide composition of oligonucleotides having the particularbase sequence and length, which composition is chirally controlled inthat it is enriched, relative to a substantially racemic preparation ofoligonucleotides having the particular base sequence and length, foroligonucleotides of a single oligonucleotide type characterized by:

-   -   1) the particular base sequence and length;    -   2) a particular pattern of backbone linkages; and    -   3) a particular pattern of backbone chiral centers,        the contacting being performed under conditions so that cleavage        of the nucleic acid polymer occurs.

In some embodiments, a provided chirally controlled oligonucleotidecomposition reduces the number of cleavage sites within the targetsequence. In some embodiments, a provided chirally controlledoligonucleotide composition provides single-site cleavage within thetarget sequence. In some embodiments, a chirally controlledoligonucleotide composition provides enhanced cleavage rate at acleavage site within the target sequence. In some embodiments, achirally controlled oligonucleotide composition provides enhancedefficiency at a cleavage site within the target sequence. In someembodiments, a chirally controlled oligonucleotide composition providesincreased turn-over in cleaving a target nucleic acid polymer.

In some embodiments, cleavage occurs with a cleavage pattern differsfrom a reference cleavage pattern. In some embodiments, a referencecleavage pattern is one observed when a nucleic acid polymer iscontacted under comparable conditions with a reference oligonucleotidecomposition. In some embodiments, a reference oligonucleotidecomposition is a chirally uncontrolled (e.g., stereorandom)oligonucleotide composition of oligonucleotides that share the commonbase sequence and length of a chirally controlled oligonucleotidecomposition. In some embodiments, a reference oligonucleotidecomposition is a substantially racemic preparation of oligonucleotidesthat share the common sequence and length.

In some embodiments, a nucleic acid polymer is RNA. In some embodiments,a nucleic acid polymer is an oligonucleotide. In some embodiments, anucleic acid polymer is an RNA oligonucleotide. In some embodiments, anucleic acid polymer is a transcript. In some embodiments,oligonucleotides of a provided chirally controlled oligonucleotidecomposition form duplexes with a nucleic acid polymer to be cleaved.

In some embodiments, a nucleic acid polymer is cleaved by an enzyme. Insome embodiments, an enzyme cleaves a duplex formed by a nucleic acidpolymer. In some embodiments, an enzyme is RNase H. In some embodiments,an enzyme is Dicer. In some embodiments, an enzyme is an Argonauteprotein. In some embodiments, an enzyme is Ago2. In some embodiments, anenzyme is within a protein complex. An exemplary protein complex isRNA-induced silencing complex (RISC).

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprising oligonucleotides with a common pattern ofbackbone chiral centers provides unexpectedly high selectivity so thatnucleic acid polymers that have only small sequence variations within atarget region can be selectively targeted. In some embodiments, anucleic acid polymer is a transcript from an allele. In someembodiments, transcripts from different alleles can be selectivelytargeted by provided chirally controlled oligonucleotide compositions.

In some embodiments, provided chirally controlled oligonucleotidecompositions and methods thereof enables precise control of cleavagesites within a target sequence. In some embodiments, a cleavage site isaround a sequence of RpSpSp backbone chiral centers. In someembodiments, a cleavage site is upstream of and near a sequence ofRpSpSp backbone chiral centers. In some embodiments, a cleavage site iswithin 5 base pairs upstream of a sequence of RpSpSp backbone chiralcenters. In some embodiments, a cleavage site is within 4 base pairsupstream of a sequence of RpSpSp backbone chiral centers. In someembodiments, a cleavage site is within 3 base pairs upstream of asequence of RpSpSp backbone chiral centers. In some embodiments, acleavage site is within 2 base pairs upstream of a sequence of RpSpSpbackbone chiral centers. In some embodiments, a cleavage site is within1 base pair upstream of a sequence of RpSpSp backbone chiral centers. Insome embodiments, a cleavage site is downstream of and near a sequenceof RpSpSp backbone chiral centers. In some embodiments, a cleavage siteis within 5 base pairs downstream of a sequence of RpSpSp backbonechiral centers. In some embodiments, a cleavage site is within 4 basepairs downstream of a sequence of RpSpSp backbone chiral centers. Insome embodiments, a cleavage site is within 3 base pairs downstream of asequence of RpSpSp backbone chiral centers. In some embodiments, acleavage site is within 2 base pairs downstream of a sequence of RpSpSpbackbone chiral centers. In some embodiments, a cleavage site is within1 base pair downstream of a sequence of RpSpSp backbone chiral centers.Among other things, the present invention therefore provides control ofcleavage sites with in a target sequence. In some embodiments, anexemplary cleavage is depicted in FIG. 21. In some embodiments, cleavagedepicted in FIG. 21 is designated as cleavage at a site two base pairsdownstream a sequence of RpSpSp backbone chiral centers. As extensivelydescribed in the present disclosure, a sequence of RpSpSp backbonechiral centers can be found in a single or repeating units of(Np)m(Rp)n(Sp)t, (Np)t(Rp)n(Sp)m, (Sp)m(Rp)n(Sp)t, (Sp)t(Rp)n(Sp)m,(Rp)n(Sp)m, (Rp)m(Sp)n, (Sp)mRp and/or Rp(Sp)m, each of which isindependently as defined above and described herein. In someembodiments, a provided chirally controlled oligonucleotide compositioncreates a new cleavage site 2 base pairs downstream of RpSpSp backbonechiral centers in a target molecule (e.g., see FIG. 21), wherein saidnew cleavage site does not exist if a reference (e.g., chirallyuncontrolled) oligonucleotide composition is used (cannot be detected).In some embodiments, a provided chirally controlled oligonucleotidecomposition enhances cleavage at a cleavage site 2 base pairs downstreamof RpSpSp backbone chiral centers in a target molecule (e.g., see FIG.21), wherein cleavage at such a site occurs at a higher percentage thanwhen a reference (e.g., chirally uncontrolled) oligonucleotidecomposition is used. In some embodiments, cleavage at such a site by aprovided chirally controlled oligonucleotide composition is at least 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500or 1000 fold of that by a reference oligonucleotide composition (forexample, when measured by percentage of cleavage at a site). In someembodiments, a provided chirally controlled oligonucleotide compositionprovides accelerated cleavage at a cleavage site 2 base pairs downstreamof RpSpSp backbone chiral centers in a target molecule (e.g., see FIG.21), compared to when a reference (e.g., chirally uncontrolled)oligonucleotide composition is used. In some embodiments, cleavage by aprovided chirally controlled oligonucleotide composition is at least 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500or 1000 fold faster than that by a reference oligonucleotidecomposition. In some embodiments, a cleavage site of a provided chirallycontrolled oligonucleotide composition 2 base pairs downstream of RpSpSpbackbone chiral centers in a target molecule (e.g., see FIG. 21) is acleavage site when a reference (e.g., chirally uncontrolled)oligonucleotide composition is used. In some embodiments, a cleavagesite of a provided chirally controlled oligonucleotide composition 2base pairs downstream of RpSpSp backbone chiral centers in a targetmolecule (e.g., see FIG. 21) is within one base pair of a cleavage sitewhen a reference (e.g., chirally uncontrolled) oligonucleotidecomposition is used. In some embodiments, a cleavage site of a providedchirally controlled oligonucleotide composition 2 base pairs downstreamof RpSpSp backbone chiral centers in a target molecule (e.g., see FIG.21) is within 2 base pairs of a cleavage site when a reference (e.g.,chirally uncontrolled) oligonucleotide composition is used. In someembodiments, it is within 3 base pairs. In some embodiments, it iswithin 4 base pairs. In some embodiments, it is within 5 base pairs. Insome embodiments, a cleavage site of a provided chirally controlledoligonucleotide composition 2 base pairs downstream of RpSpSp backbonechiral centers in a target molecule is one of the major cleavage siteswhen a reference (e.g., chirally uncontrolled) oligonucleotidecomposition is used. In some embodiments, such a site is the cleavagesite with the highest cleavage percentage when a reference (e.g.,chirally uncontrolled) oligonucleotide composition is used. In someembodiments, a cleavage site of a provided chirally controlledoligonucleotide composition 2 base pairs downstream of RpSpSp backbonechiral centers in a target molecule is one of the cleavage sites withhigher cleavage rate when a reference (e.g., chirally uncontrolled)oligonucleotide composition is used. In some embodiments, such a site isthe cleavage site with the highest cleavage rate when a reference (e.g.,chirally uncontrolled) oligonucleotide composition is used.

In some embodiments, a provided chirally controlled oligonucleotidecomposition enhances cleavage at one or more sites, e.g., relative to areference (e.g., chirally uncontrolled/stereorandom) oligonucleotidecomposition. In some embodiments, a provided chirally controlledoligonucleotide composition selectively enhances cleavage at a singlesite relative to a reference (e.g., chirally uncontrolled/stereorandom)composition. In some embodiments, a chirally controlled oligonucleotidecomposition enhances cleavage at a site by providing a higher cleavagerate. In some embodiments, a chirally controlled oligonucleotidecomposition enhances cleavage at a site by providing a higher percentageof cleavage at said site. Percentage of cleavage at a site can bedetermined by various methods widely known and practiced in the art. Insome embodiments, percentage of cleavage at a site is determined byanalysis of cleavage products, for example, as by HPLC-MS as illustratedin FIG. 18, FIG. 19 and FIG. 30; see also exemplary cleavage maps suchas FIG. 9, FIG. 10, FIG. 11, FIG. 14, FIG. 22, FIG. 25 and FIG. 26. Insome embodiments, enhancement is relative to a reference oligonucleotidecomposition. In some embodiments, enhancement is relative to anothercleavage site. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site that is apreferred cleavage site of a reference oligonucleotide composition. Insome embodiments, a preferred cleavage site, or a group of preferredcleavage sites, is a site or sites that have relatively higherpercentage of cleavage compared to one or more other cleavage sites. Insome embodiments, preferred cleavage sites can indicate preference of anenzyme. For example, for RNase H, when a DNA oligonucleotide is used,resulting cleavage sites may indicate preference of RNase H. In someembodiments, a provided chirally controlled oligonucleotide compositionenhances cleavage at a site that is a preferred cleavage site of anenzyme. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site that is not apreferred cleavage site of a reference oligonucleotide composition. Insome embodiments, a provided chirally controlled oligonucleotidecomposition enhances cleavage at a site that is not a cleavage site of areference oligonucleotide composition, effectively creating a newcleavage site which does not exist when a reference oligonucleotidecomposition is used. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site within 5 basepairs from a targeted mutation or SNP, thereby increasing selectivecleavage of the undesired target oligonucleotide. In some embodiments, aprovided chirally controlled oligonucleotide composition enhancescleavage at a site within 4 base pairs from a targeted mutation or SNP,thereby increasing selective cleavage of the undesired targetoligonucleotide. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site within 3 basepairs from a targeted mutation or SNP, thereby increasing selectivecleavage of the undesired target oligonucleotide. In some embodiments, aprovided chirally controlled oligonucleotide composition enhancescleavage at a site within 2 base pairs from a targeted mutation or SNP,thereby increasing selective cleavage of the undesired targetoligonucleotide. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site immediatelyupstream or downstream targeted mutation or SNP, thereby increasingselective cleavage of the undesired target oligonucleotide (e.g., FIG.22, Panel D, muRNA).

In some embodiments, a provided chirally controlled oligonucleotidecomposition suppresses cleavage at one or more sites, e.g., relative toa reference (e.g., chirally uncontrolled/stereorandom) oligonucleotidecomposition. In some embodiments, a provided chirally controlledoligonucleotide composition selectively suppresses cleavage at a singlesite relative to a reference (e.g., chirally uncontrolled/stereorandom)composition. In some embodiments, a chirally controlled oligonucleotidecomposition suppresses cleavage at a site by providing a lower cleavagerate. In some embodiments, a chirally controlled oligonucleotidecomposition suppresses cleavage at a site by providing a lowerpercentage of cleavage at said site. In some embodiments, suppression isrelative to a reference oligonucleotide composition. In someembodiments, suppression is relative to another cleavage site. In someembodiments, a provided chirally controlled oligonucleotide compositionsuppresses cleavage at a site that is a preferred cleavage site of areference oligonucleotide composition. In some embodiments, a preferredcleavage site, or a group of preferred cleavage sites, is a site orsites that have relatively higher percentage of cleavage compared to oneor more other cleavage sites. In some embodiments, preferred cleavagesites can indicate preference of an enzyme. For example, for RNase H,when a DNA oligonucleotide is used, resulting cleavage sites mayindicate preference of RNase H. In some embodiments, a provided chirallycontrolled oligonucleotide composition suppresses cleavage at a sitethat is a preferred cleavage site of an enzyme. In some embodiments, aprovided chirally controlled oligonucleotide composition suppressescleavage at a site that is not a preferred cleavage site of a referenceoligonucleotide composition. In some embodiments, a provided chirallycontrolled oligonucleotide composition suppresses all cleavage sites ofa reference oligonucleotide composition. In some embodiments, a providedchirally controlled oligonucleotide composition generally enhancescleavage of target oligonucleotides. In some embodiments, a providedchirally controlled oligonucleotide composition generally suppressescleavage of non-target oligonucleotides. In some embodiments, a providedchirally controlled oligonucleotide composition enhances cleavage oftarget oligonucleotides and suppresses cleavage of non-targetoligonucleotides. Using FIG. 22, Panel D, as an example, a targetoligonucleotide for cleavage is muRNA, while a non-targetoligonucleotide is wtRNA. In a subject comprising a diseased tissuecomprising a mutation or SNP, a target oligonucleotide for cleavage canbe transcripts with a mutation or SNP, while a non-targetoligonucleotide can be normal transcripts without a mutation or SNP,such as those expressed in healthy tissues.

In some embodiments, besides patterns of backbone chiral centersdescribed herein, provided oligonucleotides optionally comprisesmodified bases, modified sugars, modified backbone linkages and anycombinations thereof. In some embodiments, a provided oligonucleotide isa unimer, altmer, blockmer, gapmer, hemimer and skipmer. In someembodiments, a provided oligonucleotide comprises one or more unimer,altmer, blockmer, gapmer, hemimer or skipmer moieties, or anycombinations thereof In some embodiments, besides patterns of backbonechiral centers herein, a provided oligonucleotide is a hemimer. In someembodiments, besides patterns of backbone chiral centers herein, aprovided oligonucleotide is a 5′-hemimer with modified sugar moieties.In some embodiments, a provided oligonucleotide is 5′-hemimer with2′-modified sugar moieties. Suitable modifications are widely known inthe art, e.g., those described in the present application. In someembodiments, a modification is 2′—F. In some embodiments, a modificationis 2′—MOE. In some embodiments, a modification is s-cEt.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of both the target allele and another allele of the        same nucleic acid sequence, transcripts of the particular allele        are suppressed at a greater level than a level of suppression        observed for another allele of the same nucleic acid sequence.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of both the target allele and another allele of the        same nucleic acid seqeunce, transcripts of the particular allele        are suppressed at a greater level than a level of suppression        observed for another allele of the same nucleic acid sequence,        the contacting being performed under conditions determined to        permit the composition to suppress transcripts of the particular        allele.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of the same target nucleic acid sequence, it shows        suppression of transcripts of the particular allele at a level        that is:    -   a) greater than when the composition is absent;    -   b) greater than a level of suppression observed for another        allele of the same nucleic acid sequence; or    -   c) both greater than when the composition is absent, and greater        than a level of suppression observed for another allele of the        same nucleic acid sequence.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of the same target nucleic acid sequence, it shows        suppression of transcripts of the particule allele at a level        that is:    -   a) greater than when the composition is absent;    -   b) greater than a level of suppression observed for another        allele of the same nucleic acid sequence; or    -   c) both greater than when the composition is absent, and greater        than a level of suppression observed for another allele of the        same nucleic acid sequence, the contacting being performed under        conditions determined to permit the composition to suppress        transcripts of the particular allele.

In some embodiments, a transcript is suppressed by cleavage of saidtranscript. In some embodiments, a specific nucleotide characteristicsequence element is in an intron. In some embodiments, a specificnucleotide characteristic sequence element is in an exon. In someembodiments, a specific nucleotide characteristic sequence element ispartially in an exon and partially in an intron. In some embodiments, aspecific nucleotide characteristic sequence element comprises a mutationthat differentiates an allele from other alleles. In some embodiments, amutation is a deletion. In some embodiments, a mutation is an insertion.In some embodiments, a mutation is a point mutation. In someembodiments, a specific nucleotide characteristic sequence elementcomprises at least one single nucleotide polymorphism (SNP) thatdifferentiates an allele from other alleles.

In some embodiments, a target nucleic acid sequence is a target gene.

In some embodiments, the present invention provides a method forallele-specific suppression of a gene whose sequence comprises at leastone single nucleotide polymorphism (SNP), comprising providing achirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is completely        complementary to a sequence found in a transcript from the first        allele but not to the corresponding sequence found in a        transcript from the second allele, wherein the sequence found in        the transcripts comprises a SNP site;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers;        wherein the transcript from the first allele is suppressed at        least five folds more than that from the second allele.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of both the target allele and another allele of the        same gene,        transcripts of the particular allele are suppressed at a level        at least 2 fold greater than a level of suppression observed for        another allele of the same gene.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of both the target allele and another allele of the        same gene, transcripts of the particular allele are suppressed        at a level at least 2 fold greater than a level of suppression        observed for another allele of the same gene,        the contacting being performed under conditions determined to        permit the composition to suppress transcripts of the particular        allele.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system expressing        transcripts of both the target allele and another allele of the        same gene, transcripts of the particular allele are suppressed        at a level at least 2 fold greater than a level of suppression        observed for another allele of the same gene,        the contacting being performed under conditions determined to        permit the composition to suppress expression of the particular        allele.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in thatwhen it is contacted with a system expressing transcripts        of the target gene, it shows suppression of expression of        transcripts of the particular allele at a level that is:    -   a) at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent;    -   b) at least 2 fold greater than a level of suppression observed        for another allele of the same gene; or    -   c) both at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent, and at        least 2 fold greater than a level of suppression observed for        another allele of the same gene.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system expressing        transcripts of the target gene, it shows suppression of        expression of transcripts of the particular allele at a level        that is:    -   a) at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent;    -   b) at least 2 fold greater than a level of suppression observed        for another allele of the same gene; or    -   c) both at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent, and at        least 2 fold greater than a level of suppression observed for        another allele of the same gene, the contacting being performed        under conditions determined to permit the composition to        suppress transcripts of the particular allele.

In some embodiments, the present invention provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system expressing        transcripts of the target gene, it shows suppression of        expression of transcripts of the particular allele at a level        that is:    -   a) at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent;    -   b) at least 2 fold greater than a level of suppression observed        for another allele of the same gene; or    -   c) both at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent, and at        least 2 fold greater than a level of suppression observed for        another allele of the same gene, the contacting being performed        under conditions determined to permit the composition to        suppress expression of the particular allele.

In some embodiments, suppression of transcripts of a particular alleleis at a level that is greater than when the composition is absent. Insome embodiments, suppression of transcripts of a particular allele isat a level that is at least 1.1 fold relative to when the composition isabsent, in that transcripts from the particular allele are detected inamounts that are at least 1.1 fold lower when the composition is presentrelative to when it is absent. In some embodiments, a level is at least1.2 fold. In some embodiments, a level is at least 1.3 fold. In someembodiments, a level is at least 1.4 fold. In some embodiments, a levelis at least 1.5 fold. In some embodiments, a level is at least 1.6 fold.In some embodiments, a level is at least 1.7 fold. In some embodiments,a level is at least 1.8 fold. In some embodiments, a level is at least1.9 fold. In some embodiments, a level is at least 2 fold. In someembodiments, a level is at least 3 fold. In some embodiments, a level isat least 4 fold. In some embodiments, a level is at least 5 fold. Insome embodiments, a level is at least 6 fold. In some embodiments, alevel is at least 7 fold. In some embodiments, a level is at least 8fold. In some embodiments, a level is at least 9 fold. In someembodiments, a level is at least 10 fold. In some embodiments, a levelis at least 11 fold. In some embodiments, a level is at least 12 fold.In some embodiments, a level is at least 13 fold. In some embodiments, alevel is at least 14 fold. In some embodiments, a level is at least 15fold. In some embodiments, a level is at least 20 fold. In someembodiments, a level is at least 30 fold. In some embodiments, a levelis at least 40 fold. In some embodiments, a level is at least 50 fold.In some embodiments, a level is at least 75 fold. In some embodiments, alevel is at least 100 fold. In some embodiments, a level is at least 150fold. In some embodiments, a level is at least 200 fold. In someembodiments, a level is at least 300 fold. In some embodiments, a levelis at least 400 fold. In some embodiments, a level is at least 500 fold.In some embodiments, a level is at least 750 fold. In some embodiments,a level is at least 1000 fold. In some embodiments, a level is at least5000 fold.

In some embodiments, suppression of transcripts of a particular alleleis at a level that is greater than a level of suppression observed foranother allele of the same nucleic acid sequence. In some embodiments,suppression of transcripts of a particular allele is at a level that isat least 1.1 fold greater than a level of suppression observed foranother allele of the same nucleic acid sequence. In some embodiments, alevel is at least 1.2 fold. In some embodiments, a level is at least 1.3fold. In some embodiments, a level is at least 1.4 fold. In someembodiments, a level is at least 1.5 fold. In some embodiments, a levelis at least 1.6 fold. In some embodiments, a level is at least 1.7 fold.In some embodiments, a level is at least 1.8 fold. In some embodiments,a level is at least 1.9 fold. In some embodiments, a level is at least 2fold. In some embodiments, a level is at least 3 fold. In someembodiments, a level is at least 4 fold. In some embodiments, a level isat least 5 fold. In some embodiments, a level is at least 6 fold. Insome embodiments, a level is at least 7 fold. In some embodiments, alevel is at least 8 fold. In some embodiments, a level is at least 9fold. In some embodiments, a level is at least 10 fold. In someembodiments, a level is at least 11 fold. In some embodiments, a levelis at least 12 fold. In some embodiments, a level is at least 13 fold.In some embodiments, a level is at least 14 fold. In some embodiments, alevel is at least 15 fold. In some embodiments, a level is at least 20fold. In some embodiments, a level is at least 30 fold. In someembodiments, a level is at least 40 fold. In some embodiments, a levelis at least 50 fold. In some embodiments, a level is at least 75 fold.In some embodiments, a level is at least 100 fold. In some embodiments,a level is at least 150 fold. In some embodiments, a level is at least200 fold. In some embodiments, a level is at least 300 fold. In someembodiments, a level is at least 400 fold. In some embodiments, a levelis at least 500 fold. In some embodiments, a level is at least 750 fold.In some embodiments, a level is at least 1000 fold. In some embodiments,a level is at least 5000 fold.

In some embodiments, suppression of transcripts of a particular alleleis at a level that is greater than when the composition is absent, andat a level that is greater than a level of suppression observed foranother allele of the same nucleic acid sequence. In some embodiments,suppression of transcripts of a particular allele is at a level that isat least 1.1 fold relative to when the composition is absent, and atleast 1.1 fold greater than a level of suppression observed for anotherallele of the same nucleic acid sequence. In some embodiments, each foldis independently as described above.

In some embodiments, a system is a composition comprising a transcript.In some embodiments, a system is a composition comprising transcriptsfrom different alleles. In some embodiments, a system can be in vivo orin vitro, and in either way can comprise one or more cells, tissues,organs or organisms. In some embodiments, a system comprises one or morecells. In some embodiments, a system comprises one or more tissues. Insome embodiments, a system comprises one or more organs. In someembodiments, a system comprises one or more organisms. In someembodiments, a system is a subject.

In some embodiments, suppression of a transcript, or suppression ofexpression of an allele from which a transcript is transcribed, can bemeasured in in vitro assay. In some embodiments, a sequence from atranscript and comprising a specific nucleotide characteristic sequenceelement is usned in assays instead of the full-length transcript. Insome embodiments, an assay is a biochemical assay. In some embodiments,an assay is a biochemical assay wherein a nucleic acid polymer, forexample, a transcript or a sequence from a transcript and comprising aspecific nucleotide characteristic sequence element, is tested forcleavage by an enzyme in the presence of a chirally controlledoligonucleotide composition.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is administered to a subject. In some embodiments, a subjectis an animal. In some embodiments, a subject is a plant. In someembodiments, a subject is a human.

In some embodiments, for allele-specific suppression of transcripts froma particular allele, transcripts are cleaved at a site near a sequencedifference, for example a mutation, within a specific nucleotidecharacteristic sequence element, which sequence differencedifferentiates transcripts from a particular allele from transcriptsfrom the other alleles. In some embodiments, transcripts are selectivelycleaved at a site near such a sequence difference. In some embodiments,transcripts are cleaved at a higher percentage at a site near such asequence difference that when a chirally uncontrolled oligonucleotidecomposition is used. In some embodiments, transcripts are cleaved at thesite of a sequence difference. In some embodiments, transcripts arecleaved only at the site of a sequence difference within a specificnucleotide characteristic sequence element. In some embodiments,transcripts are cleaved at a site within 5 base pairs downstream orupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 4 base pairs downstream or upstream a sequencedifference. In some embodiments, transcripts are cleaved at a sitewithin 3 base pairs downstream or upstream a sequence difference. Insome embodiments, transcripts are cleaved at a site within 2 base pairsdownstream or upstream a sequence difference. In some embodiments,transcripts are cleaved at a site within 1 base pair downstream orupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 5 base pairs downstream a sequence difference.In some embodiments, transcripts are cleaved at a site within 4 basepairs downstream a sequence difference. In some embodiments, transcriptsare cleaved at a site within 3 base pairs downstream a sequencedifference. In some embodiments, transcripts are cleaved at a sitewithin 2 base pairs downstream a sequence difference. In someembodiments, transcripts are cleaved at a site within 1 base pairdownstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 5 base pairs upstream a sequence difference. Insome embodiments, transcripts are cleaved at a site within 4 base pairsupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 3 base pairs upstream a sequence difference. Insome embodiments, transcripts are cleaved at a site within 2 base pairsupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 1 base pair upstream a sequence difference.Such precise control of cleavage patterns, and the resulting highlyselective suppression of transcripts from a particular allele, would notbe possible without chirally controlled oligonucleotide compositions andmethods thereof provided by Applicant in this disclosure.

In some embodiments, the present invention provides methods for treatinga subject, or preventing a disease in a subject, by specificallysuppress transcripts from a particular allele, for example, an allelethat causes or may cause a disease. In some embodiments, the presentinvention provides methods for treating a subject suffering from adisease, comprising administering to the subject a pharmaceuticalcomposition comprising a chirally controlled oligonucleotidecomposition, wherein transcripts from an allele that causes orcontributes to the disease is selectively suppressed. In someembodiments, the present invention provides methods for treating asubject suffering from a disease, comprising administering to thesubject a pharmaceutical composition comprising a chirally controlledoligonucleotide composition, wherein transcripts from an allele thatcauses the disease is selectively suppressed. In some embodiments, thepresent invention provides methods for treating a subject suffering froma disease, comprising administering to the subject a pharmaceuticalcomposition comprising a chirally controlled oligonucleotidecomposition, wherein transcripts from an allele that contributes to thedisease is selectively suppressed. In some embodiments, the presentinvention provides methods for treating a subject suffering from adisease, comprising administering to the subject a pharmaceuticalcomposition comprising a chirally controlled oligonucleotidecomposition, wherein transcripts from an allele that is related to thedisease is selectively suppressed. In some embodiments, the presentinvention provides methods for preventing a disease in a subject, byspecifically suppress transcripts from a particular allele that maycause a disease. In some embodiments, the present invention providesmethods for preventing a disease in a subject, by specifically suppresstranscripts from a particular allele that increases risk of a disease inthe subject. In some embodiments, a provided method comprisesadministering to the subject a pharmaceutical composition comprising achirally controlled oligonucleotide composition. In some embodiments, apharmaceutical composition further comprises a pharmaceutical carrier.

Diseases that involves disease-causing alleles are widely known in theart, including but not limited to those described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; U.S. patent application publicationU.S. 2013/0197061; Ostergaard et al., Nucleic Acids Research 2013,41(21), 9634-9650; and Jiang et al., Science 2013, 342, 111-114. In someembodiments, a disease is Huntington's disease. In some embodiments, adisease is human hypertrophic cardiomyopathy (HCM). In some embodiments,a disease is dilated cardiomyopathy. In some embodiments, adisease-causing allele is an allele of myosin heavy chains (MHC). Insome embodiments, an exemplary disease is selected from:

Target Target Disease Target gene variation Disease Target genevariation Familial Amyloid K670N- Frontotemporal Microtubule- V337MAlzheimer's precursor protein M671L dementia with associated proteindisease (APP) (Swedish parkinsonism TAU (MAPT) mutant) linked to AmyloidK670N- chromosome 17 precursor protein M671L (FTDP-17) (APP) (SwedishEhlers-Danios Procollagen type III G252V mutant) syndrome (COL3A1)Amyloid V717F (vEDS) precursor protein (London Sickle cellHemoglobin-beta E6V (APP) mutant) anemia locus (HBB) Amyloid V717IFamilial Transthyretin (TTR) V30M precursor protein (London amyloidotic(APP) mutant) polyneuropathy Preseniline 1 L392V (FAP) (PSEN1)Fibrodysplasia Activin A receptor R206H, Amyotrophic Superoxide G93Aossificans type I (ACVR1) G356D lateral dismutase progressiva Activin Areceptor R206H sclerosis (SOD1) (FOP) type 1 (ACVR1) (ALS) SuperoxideG835R Tumors Phosphoinositide-3- I633G -> A dismutase kinase, catalytic,alpha 3140A -> G (SOD1) polypeptide (PIK3CA) Slow channel AcetylcholineaS226F Spinocerebellar Ataxin-1 (ATXN1) flanking congenital receptor(AChR) ataxia type 1 region of myasthenic (SCA1) expanded syndrome CAGrepeat (SCCMS) Machado-Joseph ATAXIN3/MJD1 SNPs linked disease/spinocereto expanded bellar ataxia type CAG repeat 3 (MJD/SCA3) SpinocerebellarAtaxin-7 (ATXN7) SNP linked to ataxia type 7 expanded (SCA7) CAG repeatParkinson's Leucine-rich repeat R1441G, disease kinase 2 (LRRK2) R1441CLeucine-rich repeat G20195S kinase 2 (LRRK2) alpha-synuclein A30PHuntington Huntingtin (HTT) SNPs linked disease to expanded CAG repeatHypertrophic MYH7 R403Q cardiomyopathyIn some embodiments, exemplary targets of, and diseases that can betreated by, provided chirally controlled oligonucleotide compositionsand methods, comprises:

Disease Target gene Target variation Familial Alzheimer's Amyloidprecursor K670N-M671L disease protein (APP) (Swedish mutant) Amyloidprecursor K670N-M671L protein (APP) (Swedish mutant) Amyloid precursorV717F (London protein (APP) mutant) Amyloid precursor V717I (Londonprotein (APP) mutant) Preseniline 1 (PSEN1) L392V Amyotrophic lateralSuperoxide dismutase G93A sclerosis (ALS) (SOD1) Superoxide dismutaseG85R (SOD1) Slow channel congenital Acetylcholine receptor aS226F,aT254I, myasthenic syndrome (AChR) a5269I (SCCMS) Frontotemporaldementia Microtubule-associated V337M with parkinsonism linked proteinTAU (MAPT) to chromosome 17 (FTDP-17) Ehlers-Danlos syndrome Procollagentype III G252V (vEDS) (COL3A1) Sickle cell anemia Hemoglobin-beta locusE6V (HBB) Familial amyloidotic Transthyretin (TTR) V30M polyneuropathy(FAP) Fibrodysplasia ossificans Activin A receptor R206H, G356Dprogressiva (FOP) type I (ACVR1) Activin A receptor R206H type I (ACVR1)Tumors KRAS G12V, G12D, G13D Tumors Phosphoinositide-3- G1633A, A3140Gkinase, catalytic, alpha polypeptide (PIK3CA) Spinocerebellar ataxiaAtaxin-1 (ATXN1) SNPs linked to type 1 (SCA1) expanded CAG repeatSpinocerebellar ataxia Ataxin-7 (ATXN7) SNPs linked to type 7 (SCA7)expanded CAG repeat Spinocerebellar Ataxia Ataxin-3 (ATXN3) SNPs linkedto Type 3 (SCA3)/Machado- expanded Joseph Disease CAG repeat Parkinson'sdisease Leucine-rich repeat R1441G, R1441C kinase 2 (LRRK2) Leucine-richrepeat G20195S kinase 2 (LRRK2) Alpha-synuclein A30P, A53T, E46K (SNCA)Huntington's disease Huntingtin (HTT) SNPs linked to expanded CAG repeatHuntington's disease- JPH3 SNPs linked to like 2 expanded CTG repeatFriedreich's ataxia FXN SNPs linked to expanded GAA repeat Fragile ×mental FMR1 SNPs linked to retardation expanded syndrome/fragile × CGGrepeat tremor ataxia syndrome Myotonic Dystophy DMPK SNPs linked to(DM1) expanded CTG repeat Myotonic Dystophy ZNF9 SNPs linked to (DM2)expanded CTG repeat Spinal-Bulbar Muscular AR SNPs linked to Atrophyexpanded CAG repeat Hypertrophic MHY7 R403Q cardiomyopathy

In some embodiments, a target Huntingtin site is selected fromrs9993542_C, rs362310_C, rs362303_C, rs10488840_G, rs363125_C,rs363072_A, rs7694687_C, rs363064_C, rs363099_C, rs363088_A,rs34315806_C, rs2298967_T, rs362272_G, rs362275_C, rs362306_G,rs3775061_A, rs1006798_A, rs16843804_C, rs3121419_C, rs362271_G,rs362273_A, rs7659144_C, rs3129322_T, rs3121417_G, rs3095074_G,rs362296_C, rs108850_C, rs2024115_A, rs916171_C, rs7685686_A,rs6844859_T, rs4690073_G, rs2285086_A, rs362331_T, rs363092_C,rs3856973_G, rs4690072_T, rs7691627_G, rs2298969_A, rs2857936_C,rs6446723_T, rs762855_A, rs1263309_T, rs2798296_G, rs363096_T,rs10015979_G, rs11731237_T, rs363080_C, rs2798235_G and rs362307_T. Insome embodiments, a target Huntingtin site is selected fromrs34315806_C, rs362273_A, rs362331_T, rs363099_C, rs7685686_A,rs362306_G, rs363064_C, rs363075_G, rs2276881_G, rs362271_G, rs362303_C,rs362322_A, rs363088_A, rs6844859_T, rs3025838_C, rs363081_G,rs3025849_A, rs3121419_C, rs2298967_T, rs2298969_A, rs16843804_C,rs4690072_T, rs362310_C, rs3856973_G, and rs2285086_A. In someembodiments, a target Huntingtin site is selected from rs362331_T,rs7685686_A, rs6844859_T, rs2298969_A, rs4690072_T, rs2024115_A,rs3856973_G, rs2285086_A, rs363092_C, rs7691627_G, rs10015979_G,rs916171_C, rs6446723_T, rs11731237_T, rs362272_G, rs4690073_G, andrs363096_T. In some embodiments, a target Huntingtin site is selectedfrom rs362267, rs6844859, rs1065746, rs7685686, rs362331, rs362336,rs2024115, rs362275, rs362273, rs362272, rs3025805, rs3025806,rs35892913, rs363125, rs17781557, rs4690072, rs4690074, rs1557210,rs363088, rs362268, rs362308, rs362307, rs362306, rs362305, rs362304,rs362303, rs362302, rs363075 and rs2298969. In some embodiments, atarget Huntingtin site is selected from:

Frequency of Heterozygosity for 24 SNP Sites in the Huntingtin mRNALocation in mRNA Reference Percent Heterozygosity (Position, nt) NumberControls HD Patients ORF, exon 20 rs363075 G/A, 10.3% (G/G, G/A, 12.8%(G/G, (2822) 89.7%) 86.2%; A/A, 0.9%) ORF, exon 25 rs35892913 G/A, 10.3%(G/G, G/A, 13.0% (G/G, (3335) 89.7%) 86.1%; A/A, 0.9%) ORF, exon 25rs1065746 G/C, 0% (G/G, 100%) G/C, 0.9% (G/G, (3389) 99.1%) ORF, exon 25rs17781557 T/G, 12.9% (T/T, T/G, 1.9% (T/T, (3418) 87.1%) 98.1%) ORF,exon 29 rs4690074 C/T, 37.9% (C/C, C/T, 35.8% (C/C, (3946) 50.9%; T/T,11.2) 59.6%; T/T, 4.6%) ORF, exon 39 rs363125 C/A, 17.5% (C/C, C/A,11.0% (C/C, (5304) 79.0%; A/A, 3.5%) 87.2%; A/A, 1.8%) ORF, exon 44 exon44 G/A, 0% (G/G, 100%) G/A, 2.8% (G/G, (6150) 97.2%) ORF, exon 48rs362336 G/A, 38.7% (G/G, G/A, 37.4% (G/G, (6736) 49.6%; A/A, 11.7%)57.9%; A/A, 4.7%) ORF, exon 50 rs362331 T/C, 45.7% (T/T, T/C, 39.4%(T/T, (7070) 31.0%; C/C, 23.3%) 49.5%; C/C, 11.0%) ORF, exon 57 rs362273A/G, 40.3% (A/A, A/G, 35.2% (A/A, (7942) 48.2%; G/G, 11.4%) 60.2%; G/G,4.6%) ORF, exon 61 rs362272 G/A, 37.1% (G/G, G/A, 36.1% (G/G, (8501)51.7%; A/A, 11.2%) 59.3%; A/A, 4.6%) ORF, exon 65 rs3025806 A/T, 0%(C/C, 100%) A/T, 0% (C/C, (9053) 100%) ORF, exon 65 exon 65 G/A, 2.3%(G/G, G/A, 0% (G/G, (9175) 97.7%) 100%) ORF, exon 67 rs362308 T/C, 0%(T/T, 100%) T/C, 0% (T/T, (9523) 100%) 3′UTR, exon 67 rs362307 C/T,13.0% (C/C, C/T, 48.6% (C/C, (9633) 87.0%) 49.5%; T/T, 1.9%) 3′UTR, exon67 rs362306 G/A, 36.0% (G/G, G/A, 35.8% (G/G, (9888) 52.6%; A/A, 11.4%)59.6%; A/A, 4.6%) 3′UTR, exon 67 rs362268 C/G, 36.8% (C/C, C/G, 35.8%(C/C, (9936) 50.0%; G/G 13.2%) 59.6%; G/G, 4.6%) 3′UTR, exon 67 rs362305C/G, 20.2% (C/C, C/G, 11.9% (C/C, (9948) 78.1%; G/G 1.8%) 85.3%; G/G,2.8%) 3′UTR, exon 67 rs362304 C/A, 22.8% (C/C, C/A, 11.9% (C/C, (10060)73.7%; A/A, 3.5%) 85.3%; A/A, 2.8%) 3′UTR, exon 67 rs362303 C/T, 18.4%(C/C, C/A, 11.9% (C/C, (10095) 79.8%; T/T, 1.8%) 85.3%; T/T, 2.8%)3′UTR, exon 67 rs1557210 C/T, 0% (C/C, 100%) C/T, 0% (C/C, (10704) 100%)3′UTR, exon 67 rs362302 C/T, 4.3% (C/C, C/T, 0% (C/C, (10708) 95.7%)100%) 3′UTR, exon 67 rs3025805 G/T, 0% (G/G, 100%) G/T, 0% (G/G, (10796)100%) 3′UTR, exon 67 rs362267 C/T, 36.2% (C/C, C/T, 35.5% (C/C, (11006)52.6%; T/T, 11.2%) 59.8%; T/T, 4.7%)In some embodiments, a chirally controlled oligonucleotide compositiontargets two or more sites. In some embodiments, targeted two or moresites are selected from sited listed herein.

It is understood by a person having ordinary skill in the art thatprovided methods apply to any similar targets containing a mismatch. Insome embodiments, a mismatch is between a maternal and paternal gene.Additional exemplary targets for suppression and/or knockdown, includingallele-specific suppression and/or knockdown, can be any geneticabnormalties, e.g., mutations, related to any diseases. In someembodiments, a target, or a set of targets, is selected from geneticdeterminants of diseases, e.g., as disclosed in Xiong, et al., The humansplicing code reveals new insights into the genetic determinants ofdisease. Science Vol. 347 no. 6218 DOI: 10.1126/science.1254806. In someembodiments, a mismatch is between a mutant and a wild type.

In some embodiments, provided chirally controlled oligonucleotidecompositions and methods are used to selectively suppressoligonucleotides with a mutation in a disease. In some embodiments, adisease is cancer. In some embodiments, provided chirally controlledoligonucleotide compositions and methods are used to selectivelysuppress transcripts with mutations in cancer. In some embodiments,provided chirally controlled oligonucleotide compositions and methodsare used to suppress transcripts of KRAS. Exemplary target KRAS sitescomprises G12V ═GGU ->GUU Position 227 G->U, G12D ═GGU->GAU Position 227G->A and G13D ═GGC ->GAC Position 230 G->A.

In some embodiments, provided chirally controlled oligonucleotidecompositions and methods provide allele-specific suppression of atranscript in an organism. In some embodiments, an organism comprises atarget gene for which two or more alleles exist. For example, a subjecthas a wild type gene in its normal tissues, while the same gene ismutated in diseased tissues such as in a tumor. In some embodiments, thepresent invention provides chirally controlled oligonucleotidecompositions and methods that selectively suppress one allele, forexample, one with a mutation or SNP. In some embodiments, the presentinvention provides treatment with higher efficacy and/or low toxicity,and/or other benefits as described in the application.

In some embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of one oligonucleotide type. Insome embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of only one oligonucleotidetype. In some embodiments, provided chirally controlled oligonucleotidecompositions has oligonucleotides of only one oligonucleotide type. Insome embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of two or more oligonucleotidetypes. In some embodiments, using such compositions, provided methodscan target more than one target. In some embodiments, a chirallycontrolled oligonucleotide composition comprising two or moreoligonucleotide types targets two or more targets. In some embodiments,a chirally controlled oligonucleotide composition comprising two or moreoligonucleotide types targets two or more mismatches. In someembodiments, a single oligonucleotide type targets two or more targets,e.g., mutations. In some embodiments, a target region ofoligonucleotides of one oligonucleotide type comprises two or more“target sites” such as two mutations or SNPs.

In some embodiments, oligonucleotides in a provided chirally controlledoligonucleotide composition optionally comprise modified bases orsugars. In some embodiments, a provided chirally controlledoligonucleotide composition does not have any modified bases or sugars.In some embodiments, a provided chirally controlled oligonucleotidecomposition does not have any modified bases. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise modified bases and sugars. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise a modified base. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise a modified sugar. Modified bases and sugars foroligonucleotides are widely known in the art, including but not limitedin those described in the present disclosure. In some embodiments, amodified base is 5-mC. In some embodiments, a modified sugar is a2′-modified sugar. Suitable 2′-modification of oligonucleotide sugarsare widely known by a person having ordinary skill in the art. In someembodiments, 2′-modifications include but are not limited to 2′—OR¹,wherein R¹ is not hydrogen. In some embodiments, a 2′-modification is2′—OR¹, wherein R¹ is optionally substituted C₁-C₆ aliphatic. In someembodiments, a 2′-modification is 2′—MOE. In some embodiments, amodification is 2′-halogen. In some embodiments, a modification is 2′—F.In some embodiments, modified bases or sugars may further enhanceactivity, stability and/or selectivity of a chirally controlledoligonucleotide composition, whose common pattern of backbone chiralcenters provides unexpected activity, stability and/or selectivity.

In some embodiments, a provided chirally controlled oligonucleotidecomposition does not have any modified sugars. In some embodiments, aprovided chirally controlled oligonucleotide composition does not haveany 2′-modified sugars. In some embodiments, the present inventionsurprising found that by using chirally controlled oligonucleotidecompositions, modified sugars are not needed for stability, activity,and/or control of cleavage patterns. Furthermore, in some embodiments,the present invention surprisingly found that chirally controlledoligonucleotide compositions of oligonucleotides without modified sugarsdeliver better properties in terms of stability, activity, turn-overand/or control of cleavage patterns. For example, in some embodiments,it is surprising found that chirally controlled oligonucleotidecompositions of oligonucleotides having no modified sugars dissociatesmuch faster from cleavage products and provide significantly increasedturn-over than compositions of oligonucleotides with modified sugars.

In some embodiments, oligonucleotides of provided chirally controlledoligonucleotide compositions useful for provided methods have structuresas extensively described in the present disclosure. In some embodiments,an oligonucleotide has a wing-core-wing structure as described. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises (Sp)mRp asdescribed. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises (Sp)₂Rp. In some embodiments, the common pattern of backbonechiral centers of a provided chirally controlled oligonucleotidecomposition comprises (Sp)m(Rp)n as described. In some embodiments, thecommon pattern of backbone chiral centers of a provided chirallycontrolled oligonucleotide composition comprises (Rp)n(Sp)m asdescribed. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises Rp(Sp)m as described. In some embodiments, the common patternof backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises Rp(Sp)₂. In some embodiments, thecommon pattern of backbone chiral centers of a provided chirallycontrolled oligonucleotide composition comprises (Sp)m(Rp)n(Sp)t asdescribed. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises (Sp)mRp(Sp)t as described. In some embodiments, the commonpattern of backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises (Sp)t(Rp)n(Sp)m as described. Insome embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprises(Sp)tRp(Sp)m as described. In some embodiments, the common pattern ofbackbone chiral centers of a provided chirally controlledoligonucleotide composition comprises SpRpSpSp. In some embodiments, thecommon pattern of backbone chiral centers of a provided chirallycontrolled oligonucleotide composition comprises (Sp)₂Rp(Sp)₂. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises (Sp)₃Rp(Sp)₃.In some embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprises(Sp)₄Rp(Sp)₄. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises (Sp)tRp(Sp)₅. In some embodiments, the common pattern ofbackbone chiral centers of a provided chirally controlledoligonucleotide composition comprises SpRp(Sp)₅. In some embodiments,the common pattern of backbone chiral centers of a provided chirallycontrolled oligonucleotide composition comprises (Sp)₂Rp(Sp)₅. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises (Sp)₃Rp(Sp)₅.In some embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprises(Sp)₄Rp(Sp)₅. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises (Sp)₅Rp(Sp)₅. In some embodiments, a common pattern ofbackbone chiral centers has only one Rp, and each of the otherinternucleotidic linkages is Sp. In some embodiments, a common baselength is greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 40, 45 or 50 as described inthe present disclosure. In some embodiments, a common base length isgreater than 10. In some embodiments, a common base length is greaterthan 11. In some embodiments, a common base length is greater than 12.In some embodiments, a common base length is greater than 13. In someembodiments, a common base length is greater than 14. In someembodiments, a common base length is greater than 15. In someembodiments, a common base length is greater than 16. In someembodiments, a common base length is greater than 17. In someembodiments, a common base length is greater than 18. In someembodiments, a common base length is greater than 19. In someembodiments, a common base length is greater than 20. In someembodiments, a common base length is greater than 21. In someembodiments, a common base length is greater than 22. In someembodiments, a common base length is greater than 23. In someembodiments, a common base length is greater than 24. In someembodiments, a common base length is greater than 25. In someembodiments, a common base length is greater than 26. In someembodiments, a common base length is greater than 27. In someembodiments, a common base length is greater than 28. In someembodiments, a common base length is greater than 29. In someembodiments, a common base length is greater than 30. In someembodiments, a common base length is greater than 31. In someembodiments, a common base length is greater than 32. In someembodiments, a common base length is greater than 33. In someembodiments, a common base length is greater than 34. In someembodiments, a common base length is greater than 35.

In some embodiments, a provided chirally controlled oligonucleotidecomposition provides higher turn-over. In some embodiments, cleavageproducts from a nucleic acid polymer dissociate from oligonucleotides ofa provided chirally controlled oligonucleotide composition at a fasterrate than from oligonucleotides of a reference oligonucleotidecomposition, for example, a chirally uncontrolled oligonucleotidecomposition. In some embodiments, a provided chirally controlledoligonucleotide composition can be administered in lower unit dosage,and/or total dosage, and/or fewer doses than chirally uncontrolledoligonucleotide composition.

In some embodiments, a chirally controlled oligonucleotide compositionprovides fewer cleavage sites in the sequence of a nucleic acid polymerthat is complementary to its common base sequence or a sequence withinits common base sequence when compared to a reference oligonucleotidecomposition. In some embodiments, a chirally controlled oligonucleotidecomposition provides fewer cleavage sites in the sequence of a nucleicacid polymer that is complementary to its common base sequence. In someembodiments, a nucleic acid polymer is selectively cleaved at a singlesite within the sequence that is complimentary to the common basesequence, or a sequence within the common base sequence, of a chirallycontrolled oligonucleotide composition. In some embodiments, a chirallycontrolled oligonucleotide composition provides higher cleavagepercentage at a cleavage site within the sequence that is complimentaryto the common base sequence, or a sequence within the common basesequence, of the chirally controlled oligonucleotide composition. Insome embodiments, a chirally controlled oligonucleotide compositionprovides higher cleavage percentage at a cleavage site within thesequence that is complimentary to the common base sequence of thechirally controlled oligonucleotide composition. In some embodiments, asite having a higher cleavage percentage is a cleavage site when areference oligonucleotide composition is used. In some embodiments, asite having a higher cleavage percentage is a cleavage site that is notpresent when a reference oligonucleotide composition is used.

It is surprisingly found that with reduced number of cleavage sites inthe complimentary sequence, cleavage rate can be unexpectedly increasedand/or higher cleavage percentage can be achieved. As demonstrated inthe examples of this disclosure, provided chirally controlledoligonucleotide compositions that produce fewer cleavage sites,especially those that provide single-site cleavage, within thecomplementary sequences of target nucleic acid polymers provide muchhigher cleavage rates and much lower levels of remaining un-cleavednucleic acid polymers. Such results are in sharp contrast to generalteachings in the art in which more cleavage sites have been pursued inorder to increase the cleavage rate.

In some embodiments, a chirally controlled oligonucleotide compositionincreases cleavage rate by 1.5 fold compared to a referenceoligonucleotide composition. In some embodiments, cleavage rate isincreased by at least 2 fold. In some embodiments, cleavage rate isincreased by at least 3 fold. In some embodiments, cleavage rate isincreased by at least 4 fold. In some embodiments, cleavage rate isincreased by at least 5 fold. In some embodiments, cleavage rate isincreased by at least 6 fold. In some embodiments, cleavage rate isincreased by at least 7 fold. In some embodiments, cleavage rate isincreased by at least 8 fold. In some embodiments, cleavage rate isincreased by at least 9 fold. In some embodiments, cleavage rate isincreased by at least 10 fold. In some embodiments, cleavage rate isincreased by at least 11 fold. In some embodiments, cleavage rate isincreased by at least 12 fold. In some embodiments, cleavage rate isincreased by at least 13 fold. In some embodiments, cleavage rate isincreased by at least 14 fold. In some embodiments, cleavage rate isincreased by at least 15 fold. In some embodiments, cleavage rate isincreased by at least 20 fold. In some embodiments, cleavage rate isincreased by at least 30 fold. In some embodiments, cleavage rate isincreased by at least 40 fold. In some embodiments, cleavage rate isincreased by at least 50 fold. In some embodiments, cleavage rate isincreased by at least 60 fold. In some embodiments, cleavage rate isincreased by at least 70 fold. In some embodiments, cleavage rate isincreased by at least 80 fold. In some embodiments, cleavage rate isincreased by at least 90 fold. In some embodiments, cleavage rate isincreased by at least 100 fold. In some embodiments, cleavage rate isincreased by at least 200 fold. In some embodiments, cleavage rate isincreased by at least 300 fold. In some embodiments, cleavage rate isincreased by at least 400 fold. In some embodiments, cleavage rate isincreased by at least 500 fold. In some embodiments, cleavage rate isincreased by at least more than 500 fold.

In some embodiments, a chirally controlled oligonucleotide compositionprovides a lower level of remaining, un-cleaved target nucleic acidpolymer compared to a reference oligonucleotide composition. In someembodiments, it is 1.5 fold lower. In some embodiments, it is at least 2fold lower. In some embodiments, it is at least 3 fold lower. In someembodiments, it is at least 4 fold lower. In some embodiments, it is atleast 5 fold lower. In some embodiments, it is at least 6 fold lower. Insome embodiments, it is at least 7 fold lower. In some embodiments, itis at least 8 fold lower. In some embodiments, it is at least 9 foldlower. In some embodiments, it is at least 10 fold lower. In someembodiments, it is at least 11 fold lower. In some embodiments, it is atleast 12 fold lower. In some embodiments, it is at least 13 fold lower.In some embodiments, it is at least 14 fold lower. In some embodiments,it is at least 15 fold lower. In some embodiments, it is at least 20fold lower. In some embodiments, it is at least 30 fold lower. In someembodiments, it is at least 40 fold lower. In some embodiments, it is atleast 50 fold lower. In some embodiments, it is at least 60 fold lower.In some embodiments, it is at least 70 fold lower. In some embodiments,it is at least 80 fold lower. In some embodiments, it is at least 90fold lower. In some embodiments, it is at least 100 fold lower. In someembodiments, it is at least 200 fold lower. In some embodiments, it isat least 300 fold lower. In some embodiments, it is at least 400 foldlower. In some embodiments, it is at least 500 fold lower. In someembodiments, it is at least 1000 fold lower.

As discussed in detail herein, the present invention provides, amongother things, a chirally controlled oligonucleotide composition, meaningthat the composition contains a plurality of oligonucleotides of atleast one type. Each oligonucleotide molecule of a particular “type” iscomprised of preselected (e.g., predetermined) structural elements withrespect to: (1) base sequence; (2) pattern of backbone linkages; (3)pattern of backbone chiral centers; and (4) pattern of backboneP-modification moieties. In some embodiments, provided oligonucloetidecompositions contain oligonucleotides that are prepared in a singlesynthesis process. In some embodiments, provided compositions containoligonucloetides having more than one chiral configuration within asingle oligonucleotide molecule (e.g., where different residues alongthe oligonucleotide have different stereochemistry); in some suchembodiments, such oligonucleotides may be obtained in a single synthesisprocess, without the need for secondary conjugation steps to generateindividual oligonucleotide molecules with more than one chiralconfiguration.

Oligonucleotide compositions as provided herein can be used as agentsfor modulating a number of cellular processes and machineries, includingbut not limited to, transcription, translation, immune responses,epigenetics, etc. In addition, oligonucleotide compositions as providedherein can be used as reagents for research and/or diagnostic purposes.One of ordinary skill in the art will readily recognize that the presentinvention disclosure herein is not limited to particular use but isapplicable to any situations where the use of synthetic oligonucleitidesis desirable. Among other things, provided compositions are useful in avariety of therapeutic, diagnostic, agricultural, and/or researchapplications.

In some embodiments, provided oligonucloetide compositions compriseoligonucleotides and/or residues thereof that include one or morestructural modifications as described in detail herein. In someembodiments, provided oligonucleotide compositions compriseoligonucleoties that contain one or more nucleic acid analogs. In someembodiments, provided oligonucleotide compositions compriseoligonucleotides that contain one or more artificial nucleic acids orresidues, including but not limited to: peptide nucleic acids (PNA),Morpholino and locked nucleic acids (LNA), glycon nucleic acids (GNA),threose nucleic acids (TNA), Xeno nucleic acids (ZNA), and anycombination thereof.

In any of the embodiments, the invention is useful foroligonucleotide-based modulation of gene expression, immune response,etc. Accordingly, stereo-defined, oligonucleotide compositions of theinvention, which contain oligonucleotides of predetermined type (i.e.,which are chirally controlled, and optionally chirally pure), can beused in lieu of conventional stereo-random or chirally impurecounterparts. In some embodiments, provided compositions show enhancedintended effects and/or reduced unwanted side effects. Certainembodimetns of biological and clinical/therapeutic applications of theinvention are discussed explicitly below.

Various dosing regimens can be utilized to administer .provided chirallycontrolled oligonucleotide compositions. In some embodiments, multipleunit doses are administered, separated by periods of time. In someembodiments, a given composition has a recommended dosing regimen, whichmay involve one or more doses. In some embodiments, a dosing regimencomprises a plurality of doses each of which are separated from oneanother by a time period of the same length; in some embodiments, adosing regimen comprises a plurality of doses and at least two differenttime periods separating individual doses. In some embodiments, all doseswithin a dosing regimen are of the same unit dose amount. In someembodiments, different doses within a dosing regimen are of differentamounts. In some embodiments, a dosing regimen comprises a first dose ina first dose amount, followed by one or more additional doses in asecond dose amount different from the first dose amount. In someembodiments, a dosing regimen comprises a first dose in a first doseamount, followed by one or more additional doses in a second (orsubsequent) dose amount that is same as or different from the first dose(or another prior dose) amount. In some embodiments, a dosing regimencomprises administering at least one unit dose for at least one day. Insome embodiments, a dosing regimen comprises administering more than onedose over a time period of at least one day, and sometimes more than oneday. In some embodiments, a dosing regimen comprises administeringmultiple doses over a time period of at least week. In some embodiments,the time period is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, adosing regimen comprises administering one dose per week for more thanone week. In some embodiments, a dosing regimen comprises administeringone dose per week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, a dosingregimen comprises administering one dose every two weeks for more thantwo week period. In some embodiments, a dosing regimen comprisesadministering one dose every two weeks over a time period of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more(e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more)weeks. In some embodiments, a dosing regimen comprises administering onedose per month for one month. In some embodiments, a dosing regimencomprises administering one dose per month for more than one month. Insome embodiments, a dosing regimen comprises administering one dose permonth for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In someembodiments, a dosing regimen comprises administering one dose per weekfor about 10 weeks. In some embodiments, a dosing regimen comprisesadministering one dose per week for about 20 weeks. In some embodiments,a dosing regimen comprises administering one dose per week for about 30weeks. In some embodiments, a dosing regimen comprises administering onedose per week for 26 weeks. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that differs from that utilized for a chirally uncontrolled(e.g., stereorandom) oligonucleotide composition of the same sequence,and/or of a different chirally controlled oligonucleotide composition ofthe same sequence. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that is reduced as compared with that of a chirally uncontrolled(e.g., sterorandom) oligonucleotide composition of the same sequence inthat it achieves a lower level of total exposure over a given unit oftime, involves one or more lower unit doses, and/or includes a smallernumber of doses over a given unit of time. In some embodiments, achirally controlled oligonucleotide composition is administeredaccording to a dosing regimen that extends for a longer period of timethan does that of a chirally uncontrolled (e.g., stereorandom)oligonucleotide composition of the same sequence Without wishing to belimited by theory, Applicant notes that in some embodiments, the shorterdosing regimen, and/or longer time periods between doses, may be due tothe improved stability, bioavailability, and/or efficacy of a chirallycontrolled oligonucleotide composition. In some embodiments, a chirallycontrolled oligonucleotide composition has a longer dosing regimencompared to the corresponding chirally uncontrolled oligonucleotidecomposition. In some embodiments, a chirally controlled oligonucleotidecomposition has a shorter time period between at least two dosescompared to the corresponding chirally uncontrolled oligonucleotidecomposition. Without wishing to be limited by theory, Applicant notesthat in some embodiments longer dosing regimen, and/or shorter timeperiods between doses, may be due to the improved safety of a chirallycontrolled oligonucleotide composition.

A single dose can contain various amounts of a type of chirallycontrolled oligonucleotide, as desired suitable by the application. Insome embodiments, a single dose contains about 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more (e.g., about350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 ormore) mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 1 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 5 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 10 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 15 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 20 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 50 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 100 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 150 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 200 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 250 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 300 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a lower amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide. Insome embodiments, a chirally controlled oligonucleotide is administeredat a lower amount in a single dose, and/or in total dose, than achirally uncontrolled oligonucleotide due to improved efficacy. In someembodiments, a chirally controlled oligonucleotide is administered at ahigher amount in a single dose, and/or in total dose, than a chirallyuncontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a higher amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide dueto improved safety.

Biologically Active Oligonucleotides

A provided oligonucleotide composition as used herein may comprisesingle stranded and/or multiply stranded oligonucleotides. In someembodiments, single-stranded oligonucleotides contain self-complementaryportions that may hybridize under relevant conditions so that, as used,even single-stranded oligonucleotides may have at least partiallydouble-stranded character. In some embodiments, an oligonucleotideincluded in a provided composition is single-stranded, double-stranded,or triple-stranded. In some embodiments, an oligonucleotide included ina provided composition comprises a single-stranded portion and amultiple-stranded portion within the oligonucleotide. In someembodiments, as noted above, individual single-stranded oligonucleotidescan have double-stranded regions and single-stranded regions.

In some embodiments, provided compositions include one or moreoligonucleotides fully or partially complementary to strand of:structural genes, genes control and/or termination regions, and/orself-replicating systems such as viral or plasmid DNA. In someembodiments, provided compositions include one or more oligonucleotidesthat are or act as siRNAs or other RNA interference reagents (RNAiagents or iRNA agents), shRNA, antisense oligonucleotides, self-cleavingRNAs, ribozymes, fragment thereof and/or variants thereof (such asPeptidyl transferase 23S rRNA, RNase P, Group I and Group II introns,GIRL branching ribozymes, Leadzyme, Hairpin ribozymes, Hammerheadribozymes, HDV ribozymes, Mammalian CPEB3 ribozyme, VS ribozymes, glmSribozymes, CoTC ribozyme, etc.), microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, U1 adaptors, triplex-formingoligonucleotides, RNA activators, long non-coding RNAs, short non-codingRNAs (e.g., piRNAs), immunomodulatory oligonucleotides (such asimmunostimulatory oligonucleotides, immunoinhibitory oligonucleotides),GNA, LNA, ENA, PNA, TNA, morpholinos, G-quadruplex (RNA and DNA),antiviral oligonucleotides, and decoy oligonucleotides.

In some embodiments, provided compositions include one or more hybrid(e.g., chimeric) oligonucleotides. In the context of the presentdisclosure, the term “hybrid” broadly refers to mixed structuralcomponents of oligonucloetides. Hybrid oliogonucleotides may refer to,for example, (1) an oligonucleotide molecule having mixed classes ofnucleotides, e.g., part DNA and part RNA within the single molecule(e.g., DNA-RNA); (2) complementary pairs of nucleic acids of differentclasses, such that DNA:RNA base pairing occurs either intramolecularlyor intermolecularly; or both; (3) an oligonucleotide with two or morekinds of the backbone or internucleotide linkages.

In some embodiments, provided compositions include one or moreoligonucleotide that comprises more than one classes of nucleic acidresidues within a single molecule. For example, in any of theembodiments described herein, an oligonucleotide may comprise a DNAportion and an RNA portion. In some embodiments, an oligonucleotide maycomprise a unmodified portion and modified portion.

Provided oligonucleotide compositions can include oligonucleotidescontaining any of a variety of modifications, for example as describedherein. In some embodiments, particular modifications are selected, forexample, in light of intended use. In some embodiments, it is desirableto modify one or both strands of a double-stranded oligonucleotide (or adouble-stranded portion of a single-stranded oligonucleotie). In someembodiments, the two strands (or portions) include differentmodifications. In some embodiments, the two strands include the samemodificatinons. One of skill in the art will appreciate that the degreeand type of modifications enabled by methods of the present inventionallow for numerous permutations of modifications to be made. Exemplarysuch modifications are described herein and are not meant to belimiting.

The phrase “antisense strand” as used herein, refers to anoligonucleotide that is substantially or 100% complementary to a targetsequence of interest. The phrase “antisense strand” includes theantisense region of both oligonucleotides that are formed from twoseparate strands, as well as unimolecular oligonucleotides that arecapable of forming hairpin or dumbbell type structures. The terms“antisense strand” and “guide strand” are used interchangeably herein.

The phrase “sense strand” refers to an oligonucleotide that has the samenucleoside sequence, in whole or in part, as a target sequence such as amessenger RNA or a sequence of DNA. The terms “sense strand” and“passenger strand” are used interchangeably herein.

By “target sequence” is meant any nucleic acid sequence whose expressionor activity is to be modulated. The target nucleic acid can be DNA orRNA, such as endogenous DNA or RNA, viral DNA or viral RNA, or other RNAencoded by a gene, virus, bacteria, fungus, mammal, or plant. In someembodiments, a target sequence is associated with a disease or disorder.

By “specifically hybridizable” and “complementary” is meant that anucleic acid can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes. In reference to the nucleic molecules of the present invention,the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LITpp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA83:9373-9377;Turner et al., 1987, /. Ain. Chem. Soc. 109:3783-3785)

A percent complcmentarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” or 100% complementarity meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. Less than perfect complementarity refers to thesituation in which some, but not all, nucleoside units of two strandscan hydrogen bond with each other. “Substantial complementarity” refersto polynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, e.g., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.In some embodiments, non-target sequences differ from correspondingtarget sequences by at least 5 nucleotides.

When used as therapeutics, a provided oligonucleotide is administered asa pharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a therapeutically effective amount of a providedoligonucleotide comprising, or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable inactiveingredient selected from pharmaceutically acceptable diluents,pharmaceutically acceptable excipients, and pharmaceutically acceptablecarriers. In another embodiment, the pharmaceutical composition isformulated for intravenous injection, oral administration, buccaladministration, inhalation, nasal administration, topicaladministration, ophthalmic administration or otic administration. Infurther embodiments, the pharmaceutical composition is a tablet, a pill,a capsule, a liquid, an inhalant, a nasal spray solution, a suppository,a suspension, a gel, a colloid, a dispersion, a suspension, a solution,an emulsion, an ointment, a lotion, an eye drop or an ear drop.

Pharmaceutical Compositions

When used as therapeutics, a provided oligonucleotide or oligonucleotidecomposition described herein is administered as a pharmaceuticalcomposition. In some embodiments, the pharmaceutical compositioncomprises a therapeutically effective amount of a providedoligonucleotides, or a pharmaceutically acceptable salt thereof, and atleast one pharmaceutically acceptable inactive ingredient selected frompharmaceutically acceptable diluents, pharmaceutically acceptableexcipients, and pharmaceutically acceptable carriers. In someembodiments, the pharmaceutical composition is formulated forintravenous injection, oral administration, buccal administration,inhalation, nasal administration, topical administration, ophthalmicadministration or otic administration. In some embodiments, thepharmaceutical composition is a tablet, a pill, a capsule, a liquid, aninhalant, a nasal spray solution, a suppository, a suspension, a gel, acolloid, a dispersion, a suspension, a solution, an emulsion, anointment, a lotion, an eye drop or an ear drop.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising chirally controlled oligonucleotide, orcomposition thereof, in admixture with a pharmaceutically acceptableexcipient. One of skill in the art will recognize that thepharmaceutical compositions include the pharmaceutically acceptablesalts of the chirally controlled oligonucleotide, or compositionthereof, described above.

A variety of supramolecular nanocarriers can be used to deliver nucleicacids. Exemplary nanocarriers include, but are not limited to liposomes,cationic polymer complexes and various polymeric. Complexation ofnucleic acids with various polycations is another approach forintracellular delivery; this includes use of PEGlyated polycations,polyethyleneamine (PEI) complexes, cationic block co-polymers, anddendrimers. Several cationic nanocarriers, including PEI andpolyamidoamine dendrimers help to release contents from endosomes. Otherapproaches include use of polymeric nanoparticles, polymer micelles,quantum dots and lipoplexes.

Additional nucleic acid delivery strategies are known in addition to theexemplary delivery strategies described herein.

In therapeutic and/or diagnostic applications, the compounds of theinvention can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington, The Science andPractice of Pharmacy, (20th ed. 2000).

Provided oligonucleotides, and compositions thereof, are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from about 0.01 to about 1000 mg, from about 0.5 to about 100mg, from about 1 to about 50 mg per day, and from about 5 to about 100mg per day are examples of dosages that may be used. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington, The Science and Practice of Pharmacy (20th ed. 2000).Preferred pharmaceutically acceptable salts include, for example,acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide,hydrochloride, maleate, mesylate, napsylate, pamoate (embonate),phosphate, salicylate, succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained- low release form as is known to those skilled inthe art. Techniques for formulation and administration may be found inRemington, The Science and Practice of Pharmacy (20th ed. 2000).Suitable routes may include oral, buccal, by inhalation spray,sublingual, rectal, transdermal, vaginal, transmucosal, nasal orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articullar, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections or other modes ofdelivery.

For injection, the agents of the invention may be formulated and dilutedin aqueous solutions, such as in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the invention intodosages suitable for systemic administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection.

The compounds can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject(e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the invention may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with oligonucleotides of this invention. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the oligonucleotides of this invention to treatproliferative diseases and cancer. Examples of known chemotherapeuticagents include, but are not limited to, adriamycin, dexamethasone,vincristine, cyclophosphamide, fluorouracil, topotecan, taxol,interferons, and platinum derivatives.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples describedbelow. The following examples are intended to illustrate the benefits ofthe present invention, but do not exemplify the full scope of theinvention.

In some embodiments, the present invention provides the followingexemplary embodiments:

E1. A chirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers.        E2. The composition of example E1, wherein one or more bases are        modified.        E3. The composition of example E1, wherein none of the bases are        modified.        E4. The composition of any one of the preceding examples,        wherein the common base sequence has at least 8 bases.        E5. The composition of any one of the preceding examples,        wherein the common base sequence has at least 10 bases.        E6. The composition of any one of the preceding examples,        wherein the common base sequence has at least 15 bases.        E7. The composition of any one of the preceding examples,        wherein at least about 20% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E8. The composition of any one of the preceding examples,        wherein at least about 50% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E9. The composition of any one of the preceding examples,        wherein at least about 80% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E10. The composition of any one of the preceding examples,        wherein at least about 85% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E11. The composition of any one of the preceding examples,        wherein at least about 90% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E12. The composition of any one of the preceding examples,        wherein at least about 95% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E13. The composition of any one of the preceding examples,        wherein at least about 97% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E14. The composition of any one of the preceding examples,        wherein at least about 98% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E15. The composition of any one of the preceding examples,        wherein at least about 99% of the oligonucleotides in the        composition have the common base sequence and length, the common        pattern of backbone linkages, and the common pattern of backbone        chiral centers.        E16. The composition of any one of the preceding examples,        wherein the single oligonucleotide comprises one or more chiral,        modified phosphate linkages.        E17. The composition of any one of the preceding examples,        wherein the single oligonucleotide has a wing-core-wing        structure.        E18. The composition of any one of the preceding examples,        wherein each wing optionally contains chiral internucleotidic        linkages.        E19. The composition of any one of the preceding examples,        wherein the chiral internucleotidic linkages within each wing        independently have the same stereochemistry.        E20. The composition of any one of the preceding examples,        wherein the chiral internucleotidic linkages of both wings are        of the same stereochemistry.        E21. The composition of any one of the preceding examples,        wherein the chiral internucleotidic linkages within each wing        independently have the same stereochemistry, and the        stereochemistry of the first wing is different from that of the        second wing.        E22. The composition of any one of the preceding examples,        wherein the first wing region independently has a length of one        or more bases.        E23. The composition of any one of the preceding examples,        wherein the first wing region independently has a length of two        or more bases.        E24. The composition of any one of the preceding examples,        wherein the first wing region independently has a length of        three or more bases.        E25. The composition of any one of the preceding examples,        wherein the first wing region independently has a length of four        or more bases.        E26, The composition of any one of the preceding examples,        wherein the first wing region independently has a length of five        or more bases.        E27. The composition of any one of the preceding examples,        wherein the first wing region independently has a length of less        than eight bases.        E28. The composition of any one of the preceding examples,        wherein the second wing region independently has a length of one        or more bases.        E29. The composition of any one of the preceding examples,        wherein the second wing region independently has a length of two        or more bases.        E30. The composition of any one of the preceding examples,        wherein the second wing region independently has a length of        three or more bases,        E31. The composition of any one of the preceding examples,        wherein the second wing region independently has a length of        four or more bases.        E32. The composition of any one of the preceding examples,        wherein the second wing region independently has a length of        five or more bases.        E33. The composition of any one of the preceding examples,        wherein the second wing region independently has a length of        less than eight bases.        E34. The composition of any one of the preceding examples,        wherein the core region has a length of five or more bases.        E36. The composition of any one of the preceding examples,        wherein the core region has a length of six or more bases.        E37. The composition of any one of the preceding examples,        wherein the core region has a length of seven or more bases.        E38. The composition of any one of the preceding examples,        wherein the core region has a length of eight or more bases.        E39. The composition of any one of the preceding examples,        wherein the core region has a length of nine or more bases.        E40. The composition of any one of the preceding examples,        wherein the core region has a length of 10 or more bases.        E41. The composition of any one of the preceding examples,        wherein the core region has a length of 15 or more bases.        E42. The composition of any one of the preceding examples,        wherein the core region has repeating pattern of        internucleotidic linkage stereochemistry.        E43. The composition of any one of the preceding examples,        wherein the repeating pattern of internucleotidic linkage        stereochemistry is (Sp)m(Rp)n or (Rp)n(Sp)m, wherein each of m        and n is independently 1, 2, 3, 4, 5, 6, 7 or 8.        E44. The composition of example E43, wherein m>n.        E45. The composition of example E43 or E44, wherein n is 1.        E46. The composition of any one of the preceding examples,        wherein the core region comprises a internucleotidic linkage        stereochemistry pattern of (Sp)m(Rp)n or (Rp)n(Sp)m, wherein        each of m and n is independently 2, 3, 4, 5, 6, 7 or 8.        E47. The composition of example E46, wherein m>n.        E48. The composition of example E47, wherein n is 1.        E49. The composition of any one of the preceding examples,        wherein 50% percent or more of the chiral internucleotidic        linkages of the core region have Sp configuration.        E50. The composition of any one of the preceding examples,        wherein 60% percent or more of the chiral internucleotidic        linkages of the core region have Sp configuration.        E51. The composition of any one of the preceding examples,        wherein the core region comprises at least 2 Rp internucleotidic        linkages.        E52. The composition of any one of the preceding examples,        wherein the core region comprises at least 3 Rp internucleotidic        linkages.        E53. The composition of any one of the preceding examples,        wherein the core region comprises at least 4 Rp internucleotidic        linkages.        E54. The composition of any one of the preceding examples,        wherein the core region comprises at least 5 Rp internucleotidic        linkages.        E55. The composition of any one of the preceding examples,        wherein at least 5 backbone internucleotidic linkages are        chiral,        E56. The composition of any one of the preceding examples,        wherein each backbone internucleotidic linkage is chiral.        E57, The composition of any one of examples E1-E55, wherein at        least one backbone internucleotidic linkage is a phosphate        linkage.        E58. The composition of any one of examples E1-E16, wherein the        single oligonucleotide comprises a region in which at least one        of the first, second, third, fifth, seventh, eighteenth,        nineteenth and twentieth internucleotidic linkages is chiral.        E59. The composition of example E58, wherein at least two of the        first, second, third, fifth, seventh, eighteenth, nineteenth and        twentieth internucleotidic linkages are chiral.        E60. The composition of any one of examples E58-E59, wherein at        least three of the first, second, third, fifth, seventh,        eighteenth, nineteenth and twentieth internucleotidic linkages        are chiral.        E61. The composition of any one of examples E58-E60, wherein at        least four of the first, second, third, fifth, seventh,        eighteenth, nineteenth and twentieth internucleotidic linkages        are chiral.        E62. The composition of any one of examples E58-E61, wherein at        least one internucleotidic linkage in the region is achiral.        E63. The composition of any one of examples E58-E62, wherein at        least one internucleotidic linkage in the region is a phosphate        linkage.        E64. The composition of any one of examples E58-E63, wherein at        least 10% of the internucleotidic linkages in the region are        phosphate linkages.        E65. The composition of any one of examples E58-E64, wherein the        first internucleotidic linkage is an Sp modified        internucleotidic linkage.        E66. The composition of any one of examples E58-E64, wherein the        first internucleotidic linkage is an Rp modified        internucleotidic linkage.        E67. The composition of any one of examples E58-E66, wherein the        second internucleotidic linkage is an Sp modified        internucleotidic linkage.        E68. The composition of any one of examples E58-E66, wherein the        second internucleotidic linkage is an Rp modified        internucleotidic linkage.        E69. The composition of any one of examples E58-E68, wherein the        thrid internucleotidic linkage is an Sp modified        internucleotidic linkage.        E70. The composition of any one of examples E58-E68, wherein the        third internucleotidic linkage is an Rp modified        internucleotidic linkage.        E71. The composition of any one of examples E58-E70, wherein the        fifth internucleotidic linkage is an Sp modified        internucleotidic linkage.        E72. The composition of any one of examples E58-E70, wherein the        fifth internucleotidic linkage is an Rp modified        internucleotidic linkage.        E73. The composition of any one of examples E58-E72, wherein the        seventh internucleotidic linkage is an Sp modified        internucleotidic linkage.        E74. The composition of any one of examples E58-E72, wherein the        seventh internucleotidic linkage is an Rp modified        internucleotidic linkage.        E75. The composition of any one of examples E58-E74, wherein the        eighteenth internucleotidic linkage is an Sp modified        internucleotidic linkage.        E76. The composition of any one of examples E58-E74, wherein the        eighteenth internucleotidic linkage is an Rp modified        internucleotidic linkage.        E77. The composition of any one of examples E58-E76, wherein the        nineteenth internucleotidic linkage is an Sp modified        internucleotidic linkage.        E78. The composition of any one of examples E58-E76, wherein the        nineteenth internucleotidic linkage is an Rp modified        internucleotidic linkage.        E79. The composition of any one of examples E58-E78, wherein the        twentieth internucleotidic linkage is an Sp modified        internucleotidic linkage.        E80. The composition of any one of examples E58-E78, wherein the        twentieth internucleotidic linkage is an Rp modified        internucleotidic linkage.        E81. The composition of any one of examples E58-E80, wherein the        region has a length of 21 bases.        E82. The composition of any one of examples E58-E81, wherein the        single oligonucleotide has a length of 21 bases.        E83. The composition of any one of examples E58-E82, wherein the        chiral internucleotidic linkage is phosphorothioate.        E84. The composition of any one of the preceding examples,        wherein the chiral internucleotidic linkage has the structure of        formula I.        E85. The composition of any one of the preceding examples,        wherein the single oligonucleotide does not have 2′-OR¹ on a        sugar moiety.        E86. The composition of any one of the preceding examples,        wherein each sugar moiety does not have 2′—M0E.        E87. The composition of any one of the preceding examples,        wherein the single oligonucleotide is not (Sp, Sp, Rp, Sp, Sp,        Rp, Sp, Sp, Rp, Sp,        Sp)—d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G]—O—(SEQ ID        NO: 152) or (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,        Rp, Rp, Rp, Rp, Rp,        Rp)—Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC        (5R-(SSR)3- 5R) (SEQ ID NO: 153), wherein in the underlined        nucleotide are 2′—O—MOE modified.        E88. The composition of any one of the preceding examples,        wherein the single oligonucleotide is not an oligonucleotide        selected from:

ONT-106 (Rp)-  PCSK9 (SEQ ID uucuAGAccuGuuuuGcuudTsdT sense NO: 154)ONT-107 (Sp)-  PCSK9 (SEQ ID uucuAGAccuGuuuuGcuudTsdT sense NO: 155)ONT-108 (Rp)-  PCSK9 (SEQ ID AAGcAAAAcAGGUCuAGAAdTsdT anti- NO: 156)sense ONT-109 (Sp)-  PCSK9 (SEQ ID AAGcAAAAcAGGUCuAGAAdTsdT anti-NO: 157) sense ONT-110 (Rp, Rp)- PCSK9 (SEQ ID asAGcAAAAcAGGUCuAGAAdTsdTanti- NO: 158) sense ONT-111 (Sp, Rp)-  PCSK9 (SEQ IDasGcAAAAcAGGUCuAGAAdTsdT anti- NO: 159) sense ONT-112 (Sp, Sp)-  PCSK9(SEQ ID asGcAAAAcAGGUCuAGAAdTsdT anti- NO: 160) sense ONT-113 (Rp, Sp)- PCSK9 (SEQ ID asGcAAAAcAGGUCuAGAAdTsdT anti- NO: 161) sense whereinlower case letters represent 2′OMe RNA residues; capital lettersrepresent 2′OH RNA residues; and bolded and “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Sp))-  (SEQ ID (1) ususcsusAsGsAscscsusGsususususG NO: 162)scsususdTsdT PCSK9 (All (Rp))-  (SEQ ID (2)ususcsusAsGsAscscsusGsususususG NO: 163) scsususdTsdT PCSK9 (All (Sp))- (SEQ ID (3) usucuAsGsAsccuGsuuuuGscuusdTsdT NO: 164) PCSK9 (All (Rp))- (SEQ ID (4) usucuAsGsAsccuGsuuuuGscuusdTsdT NO: 165) PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (5)Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, NO: 166) Rp, Sp, Rp, Sp)-ususcsusAsGsAscscsusGsususususG scsususdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (6)Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, NO: 167) Sp, Rp, Sp, Rp)-ususcsusAsGsAscscsusGsususususG scsususdTsdT wherein lower case lettersrepresent 2′-OMe RNA residues; capital letters represent RNA residues; d= 2′-deoxy residues; and “s” indicates a phosphorothioate moiety;and

PCSK9 (All (Rp))- (SEQ ID  (7) AsAsGscsAsAsAsAscsAsGsGsUsCsusA NO: 168)sGsAsAsdTsdT PCSK9 (All (Sp))- (SEQ ID  (8)AsAsGscsAsAsAsAscsAsGsGsUsCsusA NO: 169) sGsAsAsdTsdT PCSK9 (All (Rp))- (SEQ ID  (9) AsAGcAAAAcsAsGsGsUsCsusAsGsAsAs NO: 170) dTsdT PCSK9(All (Sp))-  (SEQ ID (10) AsAGcAAAAcsAsGsGsUsCsusAsGsAsAs NO: 171) dTsdTPCSK9 (All (Rp))-  (SEQ ID (11) AAsGscsAsAsAsAscAGGUCuAGAAdTsdT NO: 172)PCSK9 (All (Sp))-  (SEQ ID (12) AAsGscsAsAsAsAscAGGUCuAGAAdTsdT NO: 173)PCSK9 (All (Rp))-  (SEQ ID (13) AsAsGscAsAsAsAscAsGsGsUsCsuAsGs NO: 174)AsAsdTsdT PCSK9 (All (Sp))-  (SEQ ID (14)AsAsGscAsAsAsAscAsGsGsUsCsuAsGs NO: 175) AsAsdTsdT PCSK9 (All (Rp))- (SEQ ID (15) AsAGcAAAsAscAsGsGsUsCsusAsGsAsA NO: 176) sdTsdT PCSK9(All (Sp))-  (SEQ ID (16) AsAGcAAAsAscAsGsGsUsCsusAsGsAsA NO: 177)sdTsdT PCSK9 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (17)Sp, Rp, Sp, Rp, Sp, Rp, Sp)- NO: 178) AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (18)Rp, Sp, Rp, Sp, Rp, Sp, Rp)- NO: 179) AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT wherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d = 2′-deoxy residues; “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Rp))- (SEQ ID (19) UfsusCfsusAfsgsAfscsCfsusGfsusUfsNO: 180) usUfsgsCfsusUfsdTsdT PCSK9 (All (Sp))- (SEQ ID (20)UfsusCfsusAfsgsAfscsCfsusGfsusUfs NO: 181) usUfsgsCfsusUfsdTsdT PCSK9(All (Rp))-  (SEQ ID (21) UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgC NO: 182)fsuUfsdTsdT PCSK9 (All (Sp))-  (SEQ ID (22)UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgC NO: 183) fsuUfsdTsdT PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (23)Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, NO: 184) Rp, Sp, Rp, Sp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfs usUfsgsCfsusUfsdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (24)Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, NO: 185) Sp, Rp, Sp, Rp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfs usUfsgsCfsusUfsdTsdT wherein lowercase letters represent 2′-OMe RNA residues; capital letters represent2′-F RNA residues; d = 2′-deoxy residues; and “s” indicates aphosphorothioate moiety.and

PCSK9 (All (Rp))- (SEQ ID (25) asAfsgsCfsasAfsasAfscsAfsgsGfsusCfNO: 186) susAfsgsAfsasdTsdT PCSK9 (All (Sp))- (SEQ ID (26)asAfsgsCfsasAfsasAfscsAfsgsGfsusCf NO: 187) susAfsgsAfsasdTsdT PCSK9(All (Rp))-  (SEQ ID (27) asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsg NO: 188)sAfsasdTsdT PCSK9 (All (Sp))-  (SEQ ID (28)asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsg NO: 189) sAfsasdTsdT PCSK9(All (Rp))-  (SEQ ID (29) asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsg NO: 190)AfsadTsdT PCSK9 (All (Sp))-  (SEQ ID (30)asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsg NO: 191) AfsadTsdT PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (31) Rp, Sp, Rp, Sp, Rp, Sp)-NO: 192) asAfgCfaAfasAfscAfsgsGfsusCfsusAfs gsAfsasdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (32) Sp, Rp, Sp, Rp, Sp, Rp)-NO: 193) asAfgCfaAfasAfscAfsgsGfsusCfsusAfs gsAfsasdTsdT wherein lowercase letters represent 2′-OMe RNA residues; capital letters represent2′-F RNA residues; d = 2′-deoxy residues; and “s” indicates aphosphorothioate moiety.E89. The composition of any one of the preceding examples, wherein atleast about 50% of the internucleotidic linkages are in the Spconfiguration.E90. The composition of any one of the preceding examples, wherein thecore portion comprises at least about 5 nucleotides.E91. The composition of any one of the preceding examples, wherein thecore portion comprises at least about 10 nucleotides.E92. The composition of any one of the preceding examples, wherein thecore portion comprises at least about 15 nucleotides.E93. The composition of any one of the preceding examples, wherein thecore portion comprises at least about 20 nucleotides.E94. The composition of any one of the preceding examples, wherein thecore portion comprises at least about 25 nucleotides.E95. The composition of any one of the preceding examples, wherein(Sp)m(Rp)n or (Rp)n(Sp)m is SSR.E96. The composition of any one of examples E1-E95, wherein (Sp)m(Rp)nor (Rp)n(Sp)m is RRS.E97. The composition of any one of the preceding examples, wherein arepeating pattern is a motif comprising at least about 20% of backbonechiral centers in the Sp conformation.E98. The composition of any one of the preceding examples, wherein arepeating pattern is a motif comprising at least about 50% of backbonechiral centers in the Sp conformation.E99. The composition of any one of the preceding examples, wherein arepeating pattern is a motif comprising at least about 66% of backbonechiral centers in the Sp conformation.E100. The composition of any one of the preceding examples, wherein arepeating pattern is a motif comprising at least about 75% of backbonechiral centers in the Sp conformation.E101. The composition of any one of the preceding examples, wherein arepeating pattern is a motif comprising at least about 80% of backbonechiral centers in the Sp conformation.E102. A chirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type.        E103. The composition of example E102, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence, wherein when contacted with a nucleic acid        polymer comprising the target sequence, the chirally controlled        oligonucleotide composition provides an altered cleavage pattern        than a reference cleavage pattern from a reference        oligonucleotide composition.        E104. The composition of example E103, wherein the nucleic acid        polymer is RNA, and a reference oligonucleotide composition is a        substantially racemic preparation of oligonucleotides that share        the common sequence and length.        E105. The composition of example E103, wherein the nucleic acid        polymer is RNA, and a reference oligonucleotide composition is a        chirally uncontrolled oligonucleotide composition of        oligonucleotides that share the common sequence and length.        E106. The composition of any one of examples E103-E105, wherein        the altered cleavage pattern has fewer cleavage sites than the        reference cleavage pattern.        E107. The composition of any one of examples E103-E106, wherein        the altered cleavage pattern has only one cleavage site within        the target sequence, and the reference cleavage pattern has two        or more cleavage sites within the target sequence.        E108. The composition of example E102, wherein the common base        sequence for the oligonucleotides of the single oligonucleotide        type is or comprises a sequence that is complementary to a        characteristic sequence element that defines a particular allele        of a target gene relative to other alleles of the same target        gene that exist in a population, the composition being        characterized in that, when it is contacted with a system        expressing transcripts of both the target allele and another        allele of the same gene, transcripts of the particular allele        are suppressed at a level at least 2 fold greater than a level        of suppression observed for another allele of the same gene.        E109. The composition of example E102, wherein the common base        sequence for the oligonucleotides of the single oligonucleotide        type is or comprises a sequence that is complementary to a        characteristic sequence element that defines a particular allele        of a target gene relative to other alleles of the same target        gene that exist in a population, the composition being        characterized in that, when it is contacted with a system        expressing transcripts of the target gene, it shows suppression        of expression of transcripts of the particular allele at a level        that is:    -   a) at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent;    -   b) at least 2 fold greater than a level of suppression observed        for another allele of the same gene; or    -   c) both at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent, and at        least 2 fold greater than a level of suppression observed for        another allele of the same gene.        E110. The composition of any one of examples E102-E109, wherein        oligonucleotides of the particular oligonucleotide type comprise        a modified base.        E111. The composition of any one of examples E102-E110, wherein        oligonucleotides of the particular oligonucleotide type comprise        a modified sugar.        E112. The composition of example E111, wherein the modified        sugar comprises a 2′-modification.        E113. The composition of example E112, wherein the        2′-modification is 2′—OR¹.        E114. The composition of example E113, wherein the        2′-modification is 2′—MOE.        E115. The composition of any one of examples E102-E109, wherein        oligonucleotides of the particular oligonucleotide type have no        modified base or modified sugar.        E116. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises (Sp)m(Rp)n, wherein m is 2, 3, 4,        5, 6, 7 or 8 and n is 1, 2, 3, 4, 5, 6, 7 or 8.        E117. The composition of any one of examples E102-E116, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises (Sp)mRp, wherein m is 2, 3, 4, 5,        6, 7 or 8.        E118. The composition of any one of examples E102-E117, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises (Sp)₂Rp.        E119. The composition of any one of examples E102-E118, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises repeating (Sp)m(Rp)n, wherein m        is 2, 3, 4, 5, 6, 7 or 8 and n is 1, 2, 3, 4, 5, 6, 7 or 8.        E120. The composition of any one of examples E102-E119, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises repeating (Sp)mRp, wherein m is        2, 3, 4, 5, 6, 7 or 8.        E121. The composition of any one of examples E102-E120, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises repeating (Sp)₂Rp.        E122. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises (Rp)n(Sp)m, wherein m is 2, 3, 4,        5, 6, 7 or 8 and n is 1, 2, 3, 4, 5, 6, 7 or 8.        E123. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises Rp(Sp)m, wherein m is 2, 3, 4, 5,        6, 7 or 8.        E124. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises Rp(Sp)₂.        E125. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises repeating (Rp)n(Sp)m, wherein m        is 2, 3, 4, 5, 6, 7 or 8 and n is 1, 2, 3, 4, 5, 6, 7 or 8.        E126. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises repeating Rp(Sp)m, wherein m is        2, 3, 4, 5, 6, 7 or 8.        E127. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises repeating Rp(Sp)2.        E128. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises (Np)t(Rp)n(Sp)m, wherein each n        and t is independently 1, 2, 3, 4, 5, 6, 7 or 8, m is 2, 3, 4,        5, 6, 7 or 8, and each Np is independent Rp or Sp.        E129. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises (Sp)t(Rp)n(Sp)m, wherein each n        and t is independently 1, 2, 3, 4, 5, 6, 7 or 8 and m is 2, 3,        4, 5, 6, 7 or 8.        E130. The composition of example E128 or E129, wherein n is 1.        E131. The composition of any one of examples E128-E130, wherein        t is 2, 3, 4, 5, 6, 7 or 8.        E132. The composition of any one of examples E128-E131, wherein        m is 2, 3, 4, 5, 6, 7 or 8.        E133. The composition of any one of examples E128-E131, wherein        at least one of t and m is greater than 5.        E134. The composition of any one of examples E102-E115, wherein        the pattern of backbone chiral centers of the particular        oligonucleotide type comprises SpSpRpSpSp.        E135. The composition of any one of examples E102-E134, wherein        oligonucleotides of the particularly oligonucleotide type has        only one Rp, and each of the other internucleotidic linkages is        Sp.        E136. The composition of any one of examples E102-E135, wherein        the common base length of the particular oligonucleotide type is        greater than 10.        E137. The composition of any one of examples E102-E136, wherein        each chiral internucleotidic linkage in oligonucleotides of the        particular oligonucleotide type has a structure of formula I:

E138. The composition of example E137, wherein X is S, and Y and Z areO.E139. The composition of example E137 or E138, wherein —L—R¹ is not —H.E140. The composition of example E137 or E138, wherein a structure offormula I is a phosphorothioate diester linkage.E141. The composition of any one of examples E102-E140, whereinoligonucleotides of the particular oligonucleotide type are antisenseoligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir,ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide,triplex forming oligonucleotide, aptamer or adjuvant.E142. A method for controlled cleavage of a nucleic acid polymer, themethod comprising steps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type.        E143. The method of example E142, wherein the contacting being        performed under conditions so that cleavage of the nucleic acid        polymer occurs.        E144. The method of any one of examples E142-E143, wherein the        cleavage occurs with a cleavage pattern that differs from a        reference cleavage pattern observed when the nucleic acid        polymer is contacted under comparable conditions with a        reference oligonucleotide composition.        E145. A method for altering a cleavage pattern observed when a        nucleic acid polymer whose nucleotide sequence includes a target        sequence is contacted with a reference oligonucleotide        composition that comprises oligonucleotides having a particular        base sequence and length, which particular base sequence is or        comprises a sequence that is complementary to the target        sequence, the method comprising:

contacting the nucleic acid polymer with a chirally controlledoligonucleotide composition of oligonucleotides having the particularbase sequence and length, which composition is chirally controlled inthat it is enriched, relative to a substantially racemic preparation ofoligonucleotides having the particular base sequence and length, foroligonucleotides of a single oligonucleotide type characterized by:

-   -   1) the particular base sequence and length;    -   2) a particular pattern of backbone linkages; and    -   3) a particular pattern of backbone chiral centers.        E146. The method of example E145, wherein the contacting being        performed under conditions so that cleavage of the nucleic acid        polymer occurs.        E147. The method of any one of examples E144-E146, wherein the        reference oligonucleotide composition is a substantially racemic        preparation of oligonucleotides that share the common sequence        and length.        E148. The method of any one of examples E144-E146, wherein the        reference oligonucleotide composition is a chirally uncontrolled        oligonucleotide composition of oligonucleotides that share the        common sequence and length.        E149. The method of any one of examples E144-E148, wherein the        cleavage pattern provided by the chirally controlled        oligonucleotide composition differs from a reference cleavage        pattern in that it has fewer cleavage sites within the target        sequence found in the nucleic acid polymer than the reference        cleavage pattern.        E150. The method of example E149, wherein the cleavage pattern        provided by the chirally controlled oligonucleotide composition        has a single cleavage site within the target sequence found in        the nucleic acid polymer than the reference cleavage pattern.        E151. The method of example E150, wherein the single cleavage        site is a cleavage site in the reference cleavage pattern.        E152. The method of example E150, wherein the single cleavage        site is a cleavage site not in the reference cleavage pattern.        E153. The method of any one of examples E144-E148, wherein the        cleavage pattern provided by the chirally controlled        oligonucleotide composition differs from a reference cleavage        pattern in that it increases cleavage percentage at a cleavage        site.        E154. The method of example E153, wherein the cleavage site with        increased cleavage percentage is a cleavage site in the        reference cleavage pattern.        E155. The method of example E153, wherein the cleavage site with        increased cleavage percentage is a cleavage site not in the        reference cleavage pattern.        E156. The method of any one of examples E142-E155, wherein the        chirally controlled oligonucleotide composition provides a        higher cleavage rate of the target nucleic acid polymer than a        reference oligonucleotide composition.        E157. The method of any one of examples E142-E156, where the        cleavage rate is at least 5 fold higher. E158. The method of any        one of examples E142-E157, wherein the chirally controlled        oligonucleotide composition provides a lower level of remaining        un-cleaved target nucleic acid polymer than a reference        oligonucleotide composition. E159. The method of any one of        examples E142-E158, wherein the remaining un-cleaved target        nucleic acid polymer is at least 5 fold lower. E160. The methods        of any one of examples E142-E159, wherein the cleavage products        from the nucleic acid polymer dissociate from oligonucleotides        of the particular oligonucleotide type in the chirally        controlled oligonucleotide composition at a faster rate than        from oligonucleotides of the reference oligonucleotide        composition. E161. A method for allele-specific suppression of a        transcript from a target nucleic acid sequence for which a        plurality of alleles exist within a population, each of which        contains a specific nucleotide characteristic sequence element        that defines the allele relative to other alleles of the same        target nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of both the target allele and another allele of the        same nucleic acid sequence, transcripts of the particular allele        are suppressed at a greater level than a level of suppression        observed for another allele of the same nucleic acid sequence.        E162. A method for allele-specific suppression of a transcript        from a target gene for which a plurality of alleles exist within        a population, each of which contains a specific nucleotide        characteristic sequence element that defines the allele relative        to other alleles of the same target gene, the method comprising        steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of both the target allele and another allele of the        same gene, transcripts of the particular allele are suppressed        at a level at least 2 fold greater than a level of suppression        observed for another allele of the same gene.        E163. The method of example E161 or E162, the contacting being        performed under conditions determined to permit the composition        to suppress transcripts of the particular allele.        E164. A method for allele-specific suppression of a transcript        from a target gene for which a plurality of alleles exist within        a population, each of which contains a specific nucleotide        characteristic sequence element that defines the allele relative        to other alleles of the same target gene, the method comprising        steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system expressing        transcripts of both the target allele and another allele of the        same gene, transcripts of the particular allele are suppressed        at a level at least 2 fold greater than a level of suppression        observed for another allele of the same gene.        E165. The method of example E164, wherein the contacting being        performed under conditions determined to permit the composition        to suppress expression of the particular allele.        E166. The method of any one of examples E161-E165, wherein        transcripts of the particular allele are suppressed at a level        at least 5, 10, 20, 50, 100, 200 or 500 fold greater than a        level of suppression observed for another allele of the same        gene.        E167. A method for allele-specific suppression of a transcript        from a target nucleic acid sequence for which a plurality of        alleles exist within a population, each of which contains a        specific nucleotide characteristic sequence element that defines        the allele relative to other alleles of the same target nucleic        acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system comprising        transcripts of the same target nucleic acid sequence, it shows        suppression of transcripts of the particular allele at a level        that is:    -   a) greater than when the composition is absent;    -   b) greater than a level of suppression observed for another        allele of the same nucleic acid sequence; or    -   c) both greater than when the composition is absent, and greater        than a level of suppression observed for another allele of the        same nucleic acid sequence.        E168. A method for allele-specific suppression of a transcript        from a target gene for which a plurality of alleles exist within        a population, each of which contains a specific nucleotide        characteristic sequence element that defines the allele relative        to other alleles of the same target gene, the method comprising        steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system expressing        transcripts of the target gene, it shows suppression of        expression of transcripts of the particular allele at a level        that is:    -   a) at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent;    -   b) at least 2 fold greater than a level of suppression observed        for another allele of the same gene; or    -   c) both at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent, and at        least 2 fold greater than a level of suppression observed for        another allele of the same gene.        E169. The method of example E167 or E168, the contacting being        performed under conditions determined to permit the composition        to suppress transcripts of the particular allele.        E170. A method for allele-specific suppression of a transcript        from a target gene for which a plurality of alleles exist within        a population, each of which contains a specific nucleotide        characteristic sequence element that defines the allele relative        to other alleles of the same target gene, the method comprising        steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

-   -   1) a common base sequence and length;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the same base sequence and length, for        oligonucleotides of the particular oligonucleotide type;        wherein the common base sequence for the oligonucleotides of the        particular oligonucleotide type is or comprises a sequence that        is complementary to the characteristic sequence element that        defines a particular allele, the composition being characterized        in that, when it is contacted with a system expressing        transcripts of the target gene, it shows suppression of        expression of transcripts of the particular allele at a level        that is:    -   a) at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent;    -   b) at least 2 fold greater than a level of suppression observed        for another allele of the same gene; or    -   c) both at least 2 fold in that transcripts from the particular        allele are detected in amounts that are 2 fold lower when the        composition is present relative to when it is absent, and at        least 2 fold greater than a level of suppression observed for        another allele of the same gene.        E171. The method of example E170, wherein the contacting being        performed under conditions determined to permit the composition        to suppress expression of the particular allele.        E172. The method of any one of examples E167-E171, wherein        transcripts of the particular allele are suppressed at a level        that is at least 5, 10, 20, 50, 100, 200 or 500 fold in that        transcripts from the particular allele are detected in amounts        that are 2 fold lower when the composition is present relative        to when it is absent.        E173. The method of any one of examples E167-E172, wherein        transcripts of the particular allele are suppressed at a level        that is at least 5, 10, 20, 50, 100, 200 or 500 fold greater        than a level of suppression observed for another allele of the        same gene.        E174. The method of any one of example E161-E173, wherein the        system is an in vitro or in vivo system.        E175. The method of any one of example E161-E174, wherein the        system comprises one or more cells, tissues or organs.        E176. The method of any one of example E161-E174, wherein the        system comprises one or more organisms.        E177. The method of any one of example E161-E174, wherein the        system comprises one or more subjects.        E178. The method of any one of examples E161-E177, wherein        transcripts of the particular allele are cleaved.        E179. The method of any one of examples E161-E178, wherein the        specific nucleotide characteristic sequence element is present        within an intron of the target nucleic acid sequence or gene.        E180. The method of any one of examples E161-E178, wherein the        specific nucleotide characteristic sequence element is present        within an exon of the target nucleic acid sequence or gene.        E181. The method of any one of examples E161-E178, wherein the        specific nucleotide characteristic sequence element spans an        exon and an intron of the target nucleic acid sequence or gene.        E182. The method of any one of examples E161-E181, wherein the        specific nucleotide characteristic sequence element comprises a        mutation.        E183. The method of any one of examples E161-E181, wherein the        specific nucleotide characteristic sequence element comprises a        SNP.        E184. The method of any one of example E142-E183, wherein the        chirally controlled oligonucleotide composition is administered        to a subject.        E185. The method of any one of example E142-E184, wherein the        target nucleic acid polymer or transcripts are oligonucleotides.        E186. The method of any one of example E142-E185, wherein the        target nucleic acid polymer or transcripts are RNA.        E187. The method of any one of example E142-E186, wherein        oligonucleotides of the particular oligonucleotide type in the        chirally controlled oligonucleotide composition form duplexes        with the nucleic acid polymer or transcripts.        E188. The method of any one of example E142-E187, wherein the        nucleic acid polymer or transcripts are cleaved by an enzyme.        E189. The method of any one of example E142-E188, wherein the        enzyme is RNase H.        E190. The method of any one of example E142-E188, wherein the        enzyme is an Argonaute protein or within the RNA-induced        silencing complex (RISC).        E191. The composition of any one of examples E102-E141, wherein        the oligonucleotide type is not (Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,        Rp, Sp, Sp)—d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G]—O—(SEQ        ID NO: 194) or (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,        Sp, Rp, Rp, Rp, Rp, Rp,        Rp)—Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC        (5R—(SSR)3-5R) (SEQ ID NO: 195), wherein in the underlined        nucleotide are 2′-O—MOE modified.

E192. The composition of any one of examples E102-E141, wherein theoligonucleotide has a wing-core-wing motif, wherein the pattern ofbackbone chiral centers of the core region comprises (Np)t(Rp)n(Sp)m,wherein each n and t is independently 1, 2, 3, 4, 5, 6, 7 or 8, m is 2,3, 4, 5, 6, 7 or 8, and each Np is independent Rp or Sp.

E193. The composition of any one of examples E102-E141, wherein theoligonucleotide has a wing-core-wing motif, wherein the pattern ofbackbone chiral centers of the core region comprises (Sp)t(Rp)n(Sp)m,wherein each n and t is independently 1, 2, 3, 4, 5, 6, 7 or 8, and m is2, 3, 4, 5, 6, 7 or 8.E194. The composition of any one of examples E192 or E193, wherein n is1.E195. The composition of any one of examples E192-E194, wherein m is 2.E196. The composition of any one of examples E192-E194, wherein m is 3,4, 5, 6, 7 or 8.E197. The composition of any one of examples E192-E194, wherein m is 4,5, 6, 7 or 8.E198. The composition of any one of examples E192-E194, wherein m is 5,6, 7, or 8.E199. The composition of any one of examples E192-E194, wherein m is 6,7 or 8.E200. The composition of any one of examples E192-E194, wherein m is 7or 8.E201. The composition of any one of examples E192-E194, wherein m is 8.E202. The composition of any one of examples E192-E201, wherein t is 1.E203. The composition of any one of examples E192-E201, wherein t is 2,3, 4, 5, 6, 7 or 8.E204. The composition of any one of examples E192-E201, wherein t is 3,4, 5, 6, 7 or 8.E205. The composition of any one of examples E192-E201, wherein t is 4,5, 6, 7 or 8.E206. The composition of any one of examples E192-E201, wherein t is 5,6, 7 or 8.E207. The composition of any one of examples E192-E201, wherein t is 6,7 or 8.E208. The composition of any one of examples E192-E201, wherein t is 7or 8.E209. The composition of any one of examples E192-E201, wherein t is 8.E210. The composition of any one of examples E192-E209, wherein eachwing region independently has a length of two or more bases.E211. The composition of any one of examples E192-E210, wherein allinternucleotidic linkages are chiral, modified phosphate linkages.E212. The composition of any one of examples E192-E211, wherein the coreregion has a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, ormore bases.E213. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 18 bases.E214. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E215. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E216. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E217. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E218. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E219. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E220. The composition of any one of examples E102-E141 and E192-E212,wherein the common base sequence has at least 19 bases.E221. The composition of any one of examples E102-E141 and E192-E220,wherein the oligonucleotide is not an oligonucleotide selected from:(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G]—O—(SEQ ID NO: 196) or(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp,Rp)—Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC(5R—(SSR)3-5R) (SEQ ID NO: 197), wherein in the underlined nucleotideare 2′-O—MOE modified.E222. The composition of any one of examples E102-E141 and E192-E221,wherein the oligonucleotide is not an oligonucleotide selected from:

ONT-106 (Rp)-  PCSK9 (SEQ ID uucuAGAccuGuuuuGcuudTsdT sense NO: 198)ONT-107 (Sp)-  PCSK9 (SEQ ID uucuAGAccuGuuuuGcuudTsdT sense NO: 199)ONT-108 (Rp)-  PCSK9 (SEQ ID AAGcAAAAcAGGUCuAGAAdTsdT anti- NO: 200)sense ONT-109 (Sp)-  PCSK9 (SEQ ID AAGcAAAAcAGGUCuAGAAdTsdT anti-NO: 201) sense ONT-110 (Rp, Rp)-  PCSK9 (SEQ IDasAGcAAAAcAGGUCuAGAAdTsdT anti- NO: 202) sense ONT-111 (Sp, Rp)-  PCSK9(SEQ ID asGcAAAAcAGGUCuAGAAdTsdT anti- NO: 203) sense ONT-112 (Sp, Sp)- PCSK9 (SEQ ID asGcAAAAcAGGUCuAGAAdTsdT anti- NO: 204) sense ONT-113(Rp, Sp)-  PCSK9 (SEQ ID asGcAAAAcAGGUCuAGAAdTsdT anti- NO: 205) sensewherein lower case letters represent 2′OMe RNA residues; capital lettersrepresent 2′OH RNA residues; and bolded and “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Sp))-  (SEQ ID (1) ususcsusAsGsAscscsusGsususususG NO: 206)scsususdTsdT PCSK9 (All (Rp))-  (SEQ ID (2)ususcsusAsGsAscscsusGsususususG NO: 207) scsususdTsdT PCSK9 (All (Sp))- (SEQ ID (3) usucuAsGsAsccuGsuuuuGscuusdTsdT NO: 208) PCSK9 (All (Rp))- (SEQ ID (4) usucuAsGsAsccuGsuuuuGscuusdTsdT NO: 209) PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp,   (SEQ ID (5)Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,  NO: 210) Sp, Rp, Sp, Rp, Sp)-ususcsusAsGsAscscsusGsususususG scsususdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (6) Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,NO: 211) Rp, Sp, Rp, Sp, Rp)- ususcsusAsGsAscscsusGsususususGscsususdTsdT wherein lower case letters represent 2′-OMe RNA residues;capital letters represent RNA residues; d = 2′-deoxy residues; and “s”indicates a phosphorothioate moiety;and

PCSK9 (All (Rp))- (SEQ ID  (7) AsAsGscsAsAsAsAscsAsGsGsUsCsusAsGNO: 212) sAsAsdTsdT PCSK9 (All (Sp))- (SEQ ID  (8)AsAsGscsAsAsAsAscsAsGsGsUsCsusAsG NO: 213) sAsAsdTsdT PCSK9 (All (Rp))- (SEQ ID  (9) AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdT NO: 214) sdT PCSK9(All (Sp))-  (SEQ ID (10) AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdT NO: 215) sdTPCSK9 (All (Rp))-  (SEQ ID (11) AAsGscsAsAsAsAscAGGUCuAGAAdTsdT NO: 216)PCSK9 (All (Sp))-  (SEQ ID (12) AAsGscsAsAsAsAscAGGUCuAGAAdTsdT NO: 217)PCSK9 (All (Rp))-  (SEQ ID (13) AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAsNO: 218) AsdTsdT PCSK9 (All (Sp))-  (SEQ ID (14)AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAs NO: 219) AsdTsdT PCSK9 (All (Rp))- (SEQ ID (15) AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsd NO: 220) TsdT PCSK9(All (Sp))-  (SEQ ID (16) AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsd NO: 221)TsdT PCSK9 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (17)Sp, Rp, Sp, Rp, Sp, Rp, Sp)- NO: 222) AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (18)Rp, Sp, Rp, Sp, Rp, Sp, Rp)- NO: 223) AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT wherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d = 2′-deoxy residues; “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Rp))- (SEQ ID (19) UfsusCfsusAfsgsAfscsCfsusGfsusUfsusNO: 224) UfsgsCfsusUfsdTsdT PCSK9 (All (Sp))- (SEQ ID (20)UfsusCfsusAfsgsAfscsCfsusGfsusUfsus NO: 225) UfsgsCfsusUfsdTsdT PCSK9(All (Rp))-  (SEQ ID (21) UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfs NO: 226)uUfsdTsdT PCSK9 (All (Sp))-  (SEQ ID (22)UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfs NO: 227) uUfsdTsdT PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (23)Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, NO: 228) Sp, Rp, Sp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfsus UfsgsCfsusUfsdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (24)Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,  NO: 229) Rp, Sp, Rp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfsus UfsgsCfsusUfsdTsdT wherein lowercase letters represent 2′-OMe RNA residues; capital letters represent2′-F RNA residues; d = 2′-deoxy residues; and “s” indicates aphosphorothioate moiety;and

PCSK9 (All (Rp))- (SEQ ID (25) asAfsgsCfsasAfsasAfscsAfsgsGfsusCfNO: 230) susAfsgsAfsasdTsdT PCSK9 (All (Sp))- (SEQ ID (26)asAfsgsCfsasAfsasAfscsAfsgsGfsusCf NO: 231) susAfsgsAfsasdTsdT PCSK9(All (Rp))-  (SEQ ID (27) asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsg NO: 232)sAfsasdTsdT PCSK9 (All (Sp))-  (SEQ ID (28)asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsg NO: 233) sAfsasdTsdT PCSK9(All (Rp))-  (SEQ ID (29) asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsg NO: 234)AfsadTsdT PCSK9 (All (Sp))-  (SEQ ID (30)asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsg NO: 235) AfsadTsdT PCSK9(Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (SEQ ID (31) Rp, Sp, Rp, Sp, Rp, Sp)-NO: 236) asAfgCfaAfasAfscAfsgsGfsusCfsusAfs gsAfsasdTsdT PCSK9(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SEQ ID (32) Sp, Rp, Sp, Rp, Sp, Rp)-NO: 237) asAfgCfaAfasAfscAfsgsGfsusCfsusAfs gsAfsasdTsdTE223. The composition of any one of examples E1-E141 and E192-E222,wherein the oligonucleotide is not an oligonucleotide selected from:d[A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C] (SEQ ID NO: 54),d[C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C_(S)C_(S)C] (SEQ ID NO: 55),d[C_(S)C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C] (SEQ ID NO: 56) andd[C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C] (SEQ ID NO: 57),wherein R is Rp phosphorothioate linkage, and S is Sp phosphorothioatelinkage.E224. The composition of any one of examples E1-E141 and E192-E223,wherein the oligonucleotide is not an oligonucleotide selected from:GGA_(R)T_(S)G_(R)T_(S)T_(R) ^(m)C_(S)TCGA (SEQ ID NO: 58),GGA_(R)T_(R)G_(S)T_(S)T_(R) ^(m)C_(R)TCGA (SEQ ID NO: 59),GGA_(S)T_(S)G_(R)T_(R)T_(S) ^(m)C_(S)TCGA (SEQ ID NO: 60), wherein R isRp phosphorothioate linkage, S is Sp phosphorothioate linkage, all otherlinkages are PO, and each ^(m)C is a 5-methyl cytosine modifiednucleoside.E225. The composition of any one of examples E1-E141 and E192-E224,wherein the oligonucleotide is not an oligonucleotide selected from:T_(k)T_(k) ^(m)C_(k)AGT^(m)CATGA^(m)CT_(k)T^(m)C_(k) ^(m)C_(k) (SEQ IDNO: 61), wherein each nucleoside followed by a subscript ‘k’ indicates a(S)-cEt modification, R is Rp phosphorothioate linkage, S is Spphosphorothioate linkage, each ^(m)C is a 5-methyl cytosine modifiednucleoside, and all internucleoside linkages are phosphorothioates (PS)with stereochemistry patterns selected from RSSSRSRRRS, RSSSSSSSSS,SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS,SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR,RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS,RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS,SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.E226. The composition of any one of examples E1-E141 and E192-E225,wherein the oligonucleotide is not an oligonucleotide selected from:T_(k)T_(k) ^(m)C_(k)AGT^(m)CATGA^(m)CTT_(k) ^(m)C_(k) ^(m)C_(k) (SEQ IDNO: 62), wherein each nucleoside followed by a subscript ‘k’ indicates a(S)-cEt modification, R is Rp phosphorothioate linkage, S is Spphosphorothioate linkage, each ^(m)C is a 5-methyl cytosine modifiednucleoside and all internucleoside linkages are phosphorothioates (PS)with stereochemistry patterns selected from: RSSSRSRRRS, RSSSSSSSSS,SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS,SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR,RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS,RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS,SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.E227. The composition of any one of examples E1-E141 and E192-E226,wherein the composition comprises a predetermined level ofoligonucleotides of one oligonucleotide type characterized by a commonbase sequence, a common pattern of backbone linkages, a common patternof backbone chiral centers, and a common pattern of backbone phosphorusmodifications.E228. The composition of any one of claims E1-E141 and E192-E226,wherein the common pattern of backbone chiral centers comprises at leastabout 20% of backbone chiral centers in the Sp conformation.E229. The composition of any one of claims E1-E141 and E192-E226,wherein the common pattern of backbone chiral centers comprises at leastabout 50% of backbone chiral centers in the Sp conformation.E230. The composition of any one of claims E1-E141 and E192-E226,wherein the common pattern of backbone chiral centers comprises at leastabout 66% of backbone chiral centers in the Sp conformation.E231. The composition of any one of claims E1-E141 and E192-E226,wherein the common pattern of backbone chiral centers comprises at leastabout 75% of backbone chiral centers in the Sp conformation.E232. The composition of any one of claims E1-E141 and E192-E226,wherein the common pattern of backbone chiral centers comprises at leastabout 80% of backbone chiral centers in the Sp conformation.E233. A pharmaceutical composition, comprising a composition of any oneof claims E1-E141 and E191-E232.E234. The method of any one of examples E142-E190, wherein the chirallycontrolled oligonucleotide composition is a composition of any one ofexamples El -E101.E235. The method of any one of examples E142-E190, wherein the chirallycontrolled oligonucleotide composition is a composition of any one ofexamples E102-E141 and E191.E236. The method of any one of examples E142-E190, wherein the chirallycontrolled oligonucleotide composition is a composition of any one ofexamples E102-E141 and E191-E233.

Exemplification

The foregoing has been a description of certain nonlimiting embodimentsof the invention. Accordingly, it is to be understood that theembodiments of the invention herein described are merely illustrative ofthe application of the principles of the invention. Reference herein todetails of the illustrated embodiments is not intended to limit thescope of the claims.

EXAMPLE 1 In Vitro Metabolic Stabilities of Human Chiromersens inPreincubated Rat Whole Liver Homogenates

The present Example describes comparisons of in vitro whole rat liverhomogenate stability of Mipomersen (stereochemical mixture) withchirally controlled oligonucleotide compositions of Mipomersen(“chiromersens”). The method, among other things, is useful in screeningcompounds to predict in vivo half lives.

As is known in the art, Mipomersen (previously ISIS 301012, sold underthe trade name Kynamro) is a 20 mer oligonucleotide whose base sequenceis antisense to a portion of the apolipoprotein B gene. Mipomerseninhibits apolipoprotein B gene expression, presumably by targetgingmRNA. Mipomersen has the following structure:G*—C*—C*—U*—C*—dA—dG—dT—dC—dT—dG—dmC—dT—dT—dmC—G*—C*—A*—C*—C*   (SEQ IDNO: 65)[d=2′-deoxy, *=2′-O-(2-methoxyethyl)]with 3′→5′ phosphorothioate linkages. Thus, Mipomersen has2′-O—methoxyethyl-modified ribose residues at both ends, and deoxyriboseresidues in the middle.

Tested chirally pure Mipomersen analogs described in this Exampleincluded 3′→5′ phosphorothioate linkages. In some embodiments, testedanalogs include one or more 2′-O-(2-methoxyethyl)-modified residues; insome embodiments, tested analogs include only 2′-deoxy residues.Particular tested analogs had the structures set forth below in Tables 3and 4.

Protocol: We used the protocol reported by Geary et al.(Oligonucleotides, Volume 20, Number 6, 2010) with some modifications.

Test system: Six male Sprague-Dawley rats (Rattus norvegicus) weresupplied by Charles River Laboratories, Inc., (Hollister, Calif.), andwere received at SNBL USA.

Tissue Collection: Animals were acclimated to the study room for twodays prior to tissue collection. At the time of tissue collection,animals were anesthetized with an intraperitoneal (IP) injection ofsodium pentobarbital solution. Liver perfusion was performed using 500mL of chilled saline/animal, administered via the hepatic portal vein.After perfusion, the livers were dissected and maintained on ice. Liverswere minced into small pieces then weighed.

Liver Homogenate Preparation: The minced pieces of liver tissues weretransferred to tared 50 mL centrifuge tubes and weighed. Chilledhomogenization buffer (100 mM Tris pH 8.0, 1 mM magnesium acetate, withantibiotic-antimycotic agents) was added to each tube, such that thetube(s) contained 5 mL of buffer per gram of tissue. Using a QIAGENTissueRuptor tissue homogenizer, the liver/buffer mixture washomogenized while maintaining the tube on ice. The protein concentrationof the liver homogenate pool was determined using a Pierce BCA proteinassay. Liver homogenates were divided into 5 mL aliquots, transferred toappropriately sized labeled cryovials and stored at −60° C.

Incubation Conditions: 5 ml aliquots of frozen liver homogenate (proteinconcentration=22.48 mg/ml) were thawed and incubated at 37° C. for 24hrs. Six eppendorf tubes (2 ml) were taken for each oligomer in table 1and 450 ul of homogenate was added in each tube. 50 ul ASO (200 uM) wasadded to each tube. Immediately after mixing, 125 ul of (5X) stop buffer(2.5% IGEPAL, 0.5M NaCl, 5 mM EDTA, 50 mM Tris, pH=8.0) and 12.5 ul of20 mg/ml Proteinase K (Ambion, # AM2546) was added to one tube for 0hour time point. The remaining reaction mixtures were incubated at 37°C. with shaking at 400 rpm on VWR Incubating Microplate shaker. Afterincubation for a designated period (1, 2, 3, 4, and 5 days), eachmixture was treated with 125 ul of (5X) stop buffer (2.5% IGEPAL, 0.5MNaCl, 5 mM EDTA, 50 mM Tris, pH=8.0) and 12.5 ul of 20 mg/ml ProteinaseK (Ambion, # AM2546).

Work Up and Bioanalysis: ISIS 355868 (5′-GCGTTTGCTCTTCTTCTTGCGTTTTTT—3′(SEQ ID NO: 238)), a 27-mer oligonucleotide (underlined bases are MOEmodified) was used as the internal standard for quantitation ofchiromersens. 50 ul of internal standard (200 uM) was added to each tubefollowed by addition of 250 ul of 30% ammonium hydroxide, 800 ul ofPhenol: Chloroform: isoamyl alcohol (25:24:1). After mixing andcentrifugation at 600 rpg, the aqueous layer was evaporated on speed vacto 100 ul and loaded on Sep Pak column (C18, 1 g, WAT 036905). All theaqueous washings (2×20 ml ) of Sep pak column were tested with quick IonExchange method to ensure that no product was found there. 50% ACN (3.5ml ) was used to elute the oligonucleotide and metabolites and thecolumn was further washed with 70% CAN (3.5) ensure that there wasnothing left on the column. Five fractions were collected for eachsequence. Water wash1, 2, 3, ACN1 and 2 using Visiprep system (Sigma,part number: 57031-U).

Ion Exchange Method

Flow Time (ml/min) % A % B Curve Time 1.0 95 5 1 2 1.0 95 5 1 2 3 1.0 7525 6 3 10 1.0 35 65 6 4 10.1 1.0 95 5 6 5 12.5 1.0 95 5 1Buffer A=10 mM Tris HCl, 50%ACN, pH=8.0Buffer B=A+800 mM NaClO4Column=DNA pac 100Column Temperature 60° C.Wash method was used after each run (Described in M9-Exp21) using thesame buffers as above and 50:50 (methanol:water) in buffer line C.

Time Flow (ml/min) % A % B % C Curve Time 1.0 0 0 100 1 5.5 1.0 0 0 1001 2 5.6 1.0 100 0 0 6 3 7.5 1.0 100 0 0 6 4 7.6 1.0 95 5 0 6 5 12.5 1.095 5 0 1Acetonitrile eluate was concentrated to dryness and dissolved in 100 ulwater to be analyzed using RPHPIPC.Eluant A=10 mM Tributylammonium acetate, pH=7.0Eluant B=ACN (HPLC grade, B&J)Coulmn:XTerra MS C18, 3.5 um, 4.6×50 mm, Part number: 186000432Guard column from Phenomenex, part number: KJ0-4282Column Temperature=60° C.

HPLC Gradient:

Flow Time (ml/min) % A % B Curve 1 1.0 65 35 2 5.0 1.0 65 35 1 3 30.01.0 40 60 6 4 35.0 1.0 5 90 6 5 36.0 1.0 65 35 6 6 40.0 1.0 65 35 1For Analytical RP HPLC, 10 ul of this stock solution was added to 40 ulwater and 40 ul was injected.

TABLE 3 S. NO. Sequence Description ONT- Gs5mCs5mCs Ts5mCsAs  Mipomersen  41 GsTs5mCs TsGs5mCs TsTs5mCs  Gs5mCsAs 5mCs5mC(SEQ ID NO: 239) ONT- Gs5mCs5mCsTs5mCsAsGsTs5mCs MOE-wing-  87TsGs5mCsTsTs5mCsGs5mCsAs5m core-wing Cs5mC design - (SEQ ID NO: 240)(human) RNAse H substrate 1 5R-(SSR)₃-5R ONT- Gs5mCs5mCsTs5mCsAsGsTs5mCsAll deoxy, 154 TsGs5mCsTsTs5mCsGs5mCsAs5m (5S- Cs5mC (SSR)₃-5S)(SEQ ID NO: 241) ONT- Gs5mCsGsTsTsTsGs5mCsTs5mCs ISIS 355868  70TsTs5mCsTsTs5mCsTsTsGs5mCG internal sTsTsTsTsTsT standard for(SEQ ID NO: 242) quantitation of Mipomersen

Discussion: 2′ modifications in antisense and siRNAs are predicted tostabilize these molecules and increase their the persistence in plasmaand tissues compared with wild-type DNAs and siRNAs.

2′—MOE wing-core-wing design in Mipomersen. The first generationantisense oligonucleotides employed in the first antisense clinicaltrials had 2′-deoxy ribonucleotide residues and phosphorothioateinternucleoside linkages. Subsequently, second generation antisenseoligonucleotides were developed, which were typically of what isreferred to herein as “5-10-5 2′—MOE wing-core-wing design”, in thatfive (5) residues at each end were 2′-O-methoxyethyl (2′—MOE)-modifiedresidues and ten (10) residues in the middle were 2′-deoxyribonucleotides; the internucleotide linkages of such oligonucleotideswere phosphorothioates. Such “5-10-5 2′—MOE wing-core-wing”oligonucleotides exhibited marked improvement in potency over firstgeneration (PCT/US2005/033837). Similar wing-core-wing motifs like2-16-2, 3-14-3,4-12-4, or 5-10-5 were designed to improve the stabilityof oligonucleotides to nucleases, while at the same time maintainingenough DNA structure for RNase activity.

Chirally pure oligonucleotides. The present invention provides chirallypure oligonucleotides and demonstrates, among other things, thatselection of stereochemistry in and of itself can improveoligonucleotide stability (i.e., independent of residue modificationsuch as 2′MOE modification). Indeed, the present invention demonstratesthat chirally pure phosphorothioate oligonucleotides can provide same orbetter stability than corresponding 2′-modified stereorandomphosphorothioate compounds.

In some embodiments, tested chirally pure oligonucleotides are of thegeneral structure X—Y—X with respect to stereochemistry in that theycontain wing “X” regions (typically about 1-10 residues long) where allresidues have the same stereochemistry flanking a core “Y” region inwhich stereochemistry varies. In many embodiments, about 20-50% of thenucleotide analogs in tested such oligonucleotides are not substratesfor RNase H. The ability to control the stereochemistry ofphosphorothioates in DNA enables us to protect the oligomers fromdegradation by nucleases while maintaining the RNase active sites. Oneof these designs is ONT-154 where wings of the oligonucleotide have beenstabilized by Sp phosphorothioate chemistry with retention of few Rpphosphorothioates which are better substrates for RNase H (MolecularCell. 2007). The crystal structure of human RNase complexed with DNA/RNAduplex shows that the Phosphate-binding pocket of the enzyme makescontacts with four contiguous phosphates of DNA. The first threecontacts seem stronger than fourth one and they prefer Pro-R/Pro-R/Pro-Soxygen atoms of each of these three phosphates. Combining the stabilityadvantage coming from Sp stereochemistry with RNase H active sites,several sequences can be designed to compete with/or improve upon2′-modifications. From rat whole liver homogenate stability experimentcomparing Mipomersen (ONT-41) with our rational (chiral control) designwith and without 2′-modifications (ONT-87 and ONT-154) (Table 1 and FIG.1), it is evident that through removal of the 2′-modifications andcareful chiral control with Rp and Sp phosphorothioates, we can improvethe stability of these oligonucleotides which later affect the efficacyin vivo.

TABLE 4 Hu chiromersens studied for rat whole live homogenate stabilitySEQ ID Sequence Description Target Tm (° C.) NO: ONT-41Gs5mCs5mCs Ts5mCsAs GsTs5mCs TsGs5mCs Hu ApoB 80.7 243TsTs5mCs Gs5mCsAs 5mCs5mC ONT-75 Gs5mCs5mCsTs5mC sAsGsTs5mCsTsGs5mCsTsHu ApoB 85.0 244 Ts5mCs Gs5mCsAs5mCs5mC ONT-77 Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTs Hu ApoB 79.9 245 Ts5mCs Gs5mCsAs5mCs5mC ONT-80Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB 75.8 246 s5mCsGs5mCsAs5mCs5mC ONT-81 Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB80.7 247 s5mCs Gs5mCsAs5mCs5mC ONT-87Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB 82.4 248 s5mCsGs5mCsAs5mCs5mC ONT-88 Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB78.9 249 s5mCs Gs5mCsAs5mCs5mC ONT-89Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB 80.9 250 s5mCsGs5mCsAs5mCs5mC ONT-70 Gs5mCsGsTsTsTsGs5mCsTs5mCsTsTs5mCsTsTs ISIS 2515mCsTsTsGs5mCGsTsTsTsTsTsT 355868 internal standard

TABLE 5Mouse chiromersens studied for rat whole live homogenate stabilitySEQ ID Sequence Description Target NO: ONT-83  GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 252 ApoB ONT-82GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 253 ApoB ONT-84GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 254 ApoB ONT-85GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 255 ApoB ONT-86GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 256 ApoB

EXAMPLE 2 Exemplary Chirally Controlled siRNA Molecules

TABLE 1 Summary of Phosphodiester Polar interactions with h-Ago-2 andh-Ago-1 Science 2012 hAgo-2 Cell 2012 hAgo-2 Cell Rep 2013, h-Ago-1^(†)Phosphate* Residue Length/Å Config Phosphate Residue Length/Å ConfigPhosphate Residue Length/Å Config 2 Asn551 2.7 Pro(S) 2 Asn551 2.7Pro(S) 2 Asn549 2.7 Pro(S) Gln548 2.9 Pro(S) Gln548 3.1 Pro(S) Gln5462.9 Pro(S) Gln548 2.9 Pro(R) 2.8 Pro(R) 3 Lys566 3.1 Pro(R) 3 Lys566 2.9Pro(R) 2 Lys564 2.9 Pro(R) Arg792 3.4 Pro(R) Arg792 3.3 Pro(R) Arg7903.4 Pro(R) 3.3 Pro(R) 4 Tyr790 2.6 Pro(R) 4 Tyr790 2.8 Pro(R) 4 Try7882.7 Pro(R) Arg792 3.0 Pro(R) Arg792 2.8 Pro(R) Arg790 3.3 Pro(R) 2.8Pro(R) 3.4 Pro(S) 5 Ser798 2.7 Pro(R) 5 Ser798 2.6 Pro(R) 5 Ser796 2.5Pro(R) 2.9 Pro(R) 2.9 Pro(R) 2.8 Pro(R) Tyr804 2.8 Pro(S) Tyr804 2.5Pro(S) Tyr802 2.6 Pro(S) 6 Lys709 3.0 Pro(S) 6 Lys709 3.2 Pro(S) 6Lys707 2.8 Pro(S) Arg761 2.9 Pro(R) Arg761 2.8 Pro(R) Arg759 2.7 Pro(R)His753 2.8 Pro(R) His753 3.0 Pro(R) His751 3.0 Pro(R) 7 Arg714 2.9Pro(R) 7 Arg714 2.8 Pro(R) 7 Arg712 3.1 Pro(S) 3.0 Pro(R) 3.1 Pro(R) 3.3Pro(S) Arg761 3.0 Pro(S) Arg761 2.8 Pro(S) Arg373 3.4 Pro(R) Thr757 2.9Pro(R) 8 Arg761 2.4 Pro(S) 8 Arg759 2.2 Pro(S) Ala221 3.5 Pro(R) His7103.4 Pro(R) Ser218 2.7 Pro(R) 9 Arg351 2.2 Pro(R) 9 Arg349 3.5 Pro(R)Arg708 2.9 Pro(S) 10 Arg710 2.5 Pro(R) 10 Arg708 3.2 Pro(R) 2.9 Pro(R)18 No 21 Tyr309 2.6 Pro(S) contacts 19 Tyr311 3.1 Pro(R) Tyr314 2.6Pro(S) Arg315 2.8 Pro(R) His269 3.0 Pro(R) 20 His271 3.1 Pro(R) His3193.4 Pro(S) Tyr311 2.2 Pro(S) *Phosphate No. from S′-end ^(†)Complexedwith h-let-7 22mer

The present invention, despite teachings in the art to the contrary,recognizes that stereochemistry of internucleotidic linkages can beutilized to increase stability and activity of oligonucleotides throughchirally controlled oligonucleotide compositions. Such chirallycontrolled oligonucleotide compositions can provide much better resultsthan chirally uncontrolled oligonucleotide compositions as demonstratedin this disclosure.

There are two reported crystal structures of RNA complexed with humanArgonaute-2 protein (hAgo2): The Crystal Structure of Human Argonaute-2,Science, 2012 (PDB-4ei3); and The Structure of Human Argonaute-2 inComplex with miR-20a Cell, 2012 PDB-4f3t). In addition, there is onereported crystal structure of Let-7 RNA complexed with human Argonaute-1protein (hAgo-1): The Making of a Slicer: Activation of HumanArgonaute-1, Cell Rep. 2013 (PDB-4krf).

Based upon the information contained in these publications, it wasanticipated that some judgments could be made about advantageouspreferences for stereochemistry at the internucleotidic phosphatelinkage if the phosphodiester bonds were to be replaced byphosphorothioate diester bonds. These advantages could relate tosignificantly improved potency, stability and other pharmacologicalproperties. With this in mind, the computer program Pymol was used tolocate all polar interactions between the protein and theinternucleotidic phosphodiester linkage of the crystallized RNA for allthree structures. Polar interactions at a distance of more than 3.5 Åwere ignored.

The results of this analysis are summarized in Table 1. A particularphosphorus atom from the phosphodiester backbone on the RNA was assigneda Pro(R) or a Pro(S) configuration based upon the assumption that in thephosphorothioate diester analog the quite similar bond would be madebetween the polar group on an amino acid residue and the respectfulphosphate oxygen atom.. The sulfur substitution, instead of non-bridgingoxygen would therefore confer a unique stereochemistry (either (Sp) or(Rp) absolute configuration) on the phosphorus atom within that motif

Of note is the extraordinarily good agreement between the two structuresof hAgo-2 in complex with RNA. Also, there is an excellent agreementbetween the structures of hAgo-1 and hAgo-2 in complex with RNA,indicating that the conformation that the RNA molecule adopts is highlyconserved between these two proteins. Any conclusions or rules which areformed based upon the results of this analysis are likely, therefore, tobe valid for both protein molecules.

As can be seen, there is usually more than one polar interaction at anyone phospodiester group, with the exception of those between thephosphodiesters at phosphate positions 9 and 10 and hAgo-2 (Cell 2012)which adopt exclusively Pro(Rp) preference through bonding with Arg351and Arg710 respectively.

However, shorter distances (corresponding to stronger interactions) aswell as the number of bonds per oxygen can suggest a predominantinteraction for the Pro(Rp) or the Pro(Sp) oxygens: henceresulting inseveral interactions which are predominantly of one stereochemical typeor the other. Within this group are the interactions between thephosphodiesters at phosphate positions 2 (Sp), 3 (Rp), 4 (Rp), 6 (Rp), 8(Sp), 19 (Rp), 20 (Sp) and 21 (Sp).

Of the remaining interactions, there does not appear to be a preferencefor one particular stereochemistry to be adopted over the other, so thepreferred stereochemistry could be either (Sp) or (Rp).

Within this category are the interactions formed between thephosphodiesters at phosphate positions 5 (Rp or Sp) and 7 (Rp or Sp).

For interactions at the other phosphate backbone, there is no crystalstructure information, so stereochemistry at these positions cansimilarly be either (Rp) or (Sp) until empirical data shows otherwise.

To this end, Table 6 contains several non-limiting exemplary siRNAgeneral constructs which can be conceived to take advantage of thispreference for stereochemistry at individual phosphorothioate diestermotifs.

TABLE 6 Exemplary general siRNA constructs PS* Chirally ControlledAntisense Strand Construct 2 (Sp) (Rp) (Sp) (Rp) (Sp) (Rp) 3 (Rp) (Sp)(Rp) (Sp) (Rp) (Sp) 4 (Rp) (Sp) PO PO (Rp) (Sp) 5 (Rp) or (Sp) (Sp) or(Rp) PO PO PO PO 6 (Rp) (Sp) PO PO (Rp) (Sp) 7 (Rp) or (Sp) (Sp) or (Rp)PO PO PO PO 8 (Sp) (Rp) PO PO (Sp) (Rp) 9 (Rp) (Sp) PO PO (Rp) (Sp) 10(Rp) (Sp) PO PO (Rp) (Sp) 11 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 12(Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 13 (Rp) or (Sp) (Sp) or (Rp) PO POPO PO 14 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 15 (Rp) or (Sp) (Sp) or(Rp) PO PO PO PO 16 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 17 (Rp) or(Sp) (Sp) or (Rp) PO PO PO PO 18 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO19 (Rp) (Sp) PO PO (Rp) (Sp) 20 (Sp) (Rp) (Sp) (Rp) (Sp) (Rp) 21 (Sp)(Rp) (Sp) (Rp) (Sp) (Rp) *The number indicates the phosphate positionfrom the 5′ end of the antisense strand of the siRNA, (e.g. #2 islocated between nucleotides 1 and 2 and #21 is located betweennucleotides 20 and 21). (Sp) and (Rp) designates stereochemistry ofphosphorus atom on phosphorothioate (PS) diester internucleotidiclinkage at the indicated position. PO designates a phosphodiesterinternucleotidic linkage at the indicated position.

Exemplary siRNAs include but are not limited to siRNAs having a Spconfiguration for a chiral phosphorothioate at the 3′end and at the5′end of the antisense strand of the siRNA duplex, which confersunprecedentedly increased stability in human serum or biological fluids.That same Sp configuration for the chiral phosphorothioate at the 3′endand at the 5′end of the antisense strand of the siRNA duplex confersunprecedentedly increased biological potency caused by increasedaffitnity to the Ago2 protein leading to increased activity within theRISC RNAi silencing complex.

In one embodiment, a single chiral phosphorothioate motif is introducedindependently at each position along the antisense or sense strand ofthe siRNA molecule. For a 21 mer, this provides 80 unique sequences,with either an (Sp) or an (Rp) chirally controlled phosphorothioategroup. When duplexed independently, 1600 unique combinations of siRNAsare prepared.

siRNA Transfection of Chiral siRNA Molecules

Hep3B, or HeLa cells are reverse transfected at a density of 2.0×10⁴cells/well in 96-well plates. Transfection of siRNA is carried out withiipofectamine RNAiMax (Life Technologies, cat. No. 13778-150) using themanufacturer's protocol, except with a decreased amount ofLipofectarnine RNAiMax of 0.2 ul per well. Twelve, 1:3 siRNA duplexdilutions are created starting at 1 uM. 10 ul of 10x siRNA duplex isthen lipoplexed with a prepared mixture of 9.8 ul of serum-free mediumand 0.2 ul of Lipofectamine RNAiMax per well. After a 10-15 minuteincubation, 2.0×10⁴ cells in 80 ul of EMEM cell growing media (ATCC,30-2003) is added to bring the final volume to 100 ul per well. Twoseparate transfection events are performed for each dose.

24 hours after transfection Hep3B or HeLa cells are lysed and mRNAagainst which the siRNA is targeted is purified using MagMAX™-96 TotalRNA Isolation Kit (Life Technologies, AM1830), 15 ul of cDNA issynthesized with High Capacity cDNA Reverse Transcription Kit with RNaseInhibitor (Life Technologies, 4374967). Gene expression is evaluated byReal-Time PCR on a Lightcycler 480(Roche) using a Probes MasterMix(Roche, 04 707 494 001) according to manufacturer's protocol.

IC50s and Data Analysis

Delta delta Ct method is used to calculate values. Samples arenormalized to hGAPDH and calibrated to mock transfected and untreatedsamples. A stereo-random molecule is used as a control. The data isrepresented as a mean of 2 biological replicates using Graphpad Prism. Afour-parameter linear regression curve is fitted to the data and thebottom and top are constrained to a 0 and 100 constants respectively inorder to calculate a relative IC50.

The present Example demonstrates successful inhibition of target geneexpression using siRNA agents comprised of chirally controlledoligonucleotides as described herein. Specifically, this Exampledescribes hybridization of individual oligonucleotide strands preparedthrough chirally controlled synthesis as described herein, so thatdouble-stranded chirally controlled siRNA oligonucleotide compositionsare provided. This Example further demonstrates successful transfectionof cells with such agents and, moreover, successful inhibition of targetgene expression.

In Vitro Metabolic Stabilities of Human PCSK9 siRNA Duplexes HavingStereocontrolled Phosphorothioate Diester Linkages in Human Serum.

10 μM siRNA duplexes were incubated in 90% human serum (50 μL, Sigma,H4522) at 37° C. for 24 hours. A 0 min time point (50 μL) was preparedas well as a PBS control incubation time point (50 μL), where the 10 μMsiRNA duplex was incubated in 90% 1×PBS (50 μL at 37° C. for 24 hours.After completion of the incubation, to each time point, were added 10 μLof Stop-Solution (0.5 M NaCl, 50 mM TRIS, 5 mM EDTA, 2.5% IGEPAL),followed by 3.2 μL of Proteinase K (20 mg/mL, Ambion). The samples wereincubated at 60° C. for 20 min, and then centrifuged at 2000 rpm for 15min. The final reaction mixtures were directly analyzed in denaturingIEX HPLC (injection volume 50 μL). The ratio of integrated area at 24 hand 0 min was used to determine the % of degradation for each siRNA.

It was observed that the stereochemistry configuration of the singlephosphorothioate at position 21 (3′end) of both the antisense strand andthe sense strand of the siRNA had a crucial impact on the stability ofthe duplex upon incubation in Human Serum (FIG. 1). As illustrated inthe FIG. 1 and as determined following the integration ratio of thedegradation pattern, an (Rp, Rp) siRNA duplex exhibited a significant55.0% degradation after 24 h. The stereorandom mixture ofphosphorothioates in the stereorandom siRNA showed 25.2% degradationafter 24 h. The (Sp/Sp) siRNA showed only minor 7.3% degradation after24 h. This illustrates the drastic impact that phosphorothioatestereochemistry confers to therapeutic siRNAs. Additional exemplary datawere presented in FIG. 2, FIG. 3, FIG. 4 and FIG. 5.

It is observed that each of the stereopure constructs show differentpotency (IC₅₀ values) dependent on the position of the phosphorothioatemotif along the backbone. It is also observered that different IC₅₀values are obtained dependent upon whether the phosphorothioate motif atany single position is (Sp) or (Rp). The impact of stereochemistry uponstability is likewise dear and differentiating, using either Human Serumdescribed above, or Human Hepatic Cytosol extract or Snake VenomPhosphodiesterase, or isolated endonuclease or isolated exonuclease.

Certain design rules may be formulated based upon data obtained in theabove example. These design information can be applied for theintroduction of multiple chiral phosphorothioate linkages within theanti sense and/or sense strand of the siRNA as exemplified below. Thepresent invention recognizes that an increased amount of chiralphosphorothioate within the antisense and/or sense strand of the siRNA,introduced at the right positions and having the right stereochemistryconfiguration leads to greatly improved siRNA constructs in terms ofpotency and metabolic stability in vitro—translating into greatlypharmacologically enhanced therapeutic siRNAs.

Exemplary Chirally Controlled siRNA Oligonucleotides Targeting PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9), is an enzymeinvolved in cholesterol metabolism. PCSK9 binds to the receptor for lowdensity lipoprotein (LDL), triggering its destruction. Although LDLassociated with the receptor is also eliminated when the receptor isdestroyed, the net effect of PCSK9 binding in fact increases LDL levels,as the receptor would otherwise cycle back to the cell surface andremove more cholesterol.

Several companies are developing therapeutic agents that target PCSK9.Of particular relevance to the present disclosure, each of IsisPharmaceuticals, Santaris Pharma, and Alnylam Pharmaceuticals isdeveloping a nucleic acid agent that inhibits PCSK9. The IsisPharmaceuticals product, an antisense oligonucleotide, has been shown toincrease expression of the LDLR and decrease circulating totalcholesterol levels in mice (Graham et al “Antisense inhibition ofproprotein convertase subtilisin/kexin type 9 reduces serum LDL inhyperlipidemic mice”. J. Lipid Res. 48 (4): 763-7, April 2007). Initialclinical trials with the Alnylam Pharmaceuticals product, ALN-PCS,reveal that RNA interference offers an effective mechanism forinhibiting PCSK9 (Frank-Kamenetsky et al “Therapeutic RNAi targetingPCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterolin nonhuman primates”. Proc. Natl. Acad. Sci. U.S.A. 105 (33): 11915-20,August 2008).

In some embodiments, despite known results to the contrary, the presentinvention recognizes that phosphorothioate motifs of one stereochemicalconformation or another can be rationally designed to take advantage ofincreased potency, stability and other pharmacological qualities throughchirally controlled oligonucleotide compositions. To reinforce thisconcept, table 3 contains exemplary stereochemically pure constructsbased on an siRNA sequence which targets PCSK9 messenger RNA.

In this exemplary embodiment, a single chiral phosphorothioate motif isintroduced independently at each position along the antisense or sensestrand of the siRNA molecule. For a 21 mer, this provides 80 uniquesequences, with either an (Sp) or an (Rp) chirally controlledphosphorothioate group. When duplexed independently, 1600 uniquecombinations of siRNAs are prepared.

In other exemplary embodiments, a single chiral phosphorothioate motifis introduced independently at each position along the antisense orsense strand of the siRNA molecule, while a 3′-(Sp) phosphorothioatelinkage is conserved. For a 21 mer, this provides another additional 80unique sequences, with either an (Sp) or an (Rp) chirally controlledphosphorothioate group. When duplexed independently, 1600 uniquecombinations of siRNAs are prepared.

In other exemplary embodiments, multiple chiral phosphorothioate motifsare introduced independently at several positions along the antisense orsense strand of the siRNA molecule, following the codes described inTable 7, while a 3′-(Sp) phosphorothioate linkage is conserved.

TABLE 7 Example of PCSK-9 Sense and Antisense RNAs SEQ IDPCSK9 siRNA Sense Strands NO: PCSK9 (1) (Rp)-uucuAGAccuGuuuuGcuudTsdT257 PCSK9 (2) (Sp)-uucuAGAccuGuuuuGcuudTsdT 258 PCSK9 (3)(Rp)-uucuAGAccuGuuuuGcuusdTdT 259 PCSK9 (4)(Sp)-uucuAGAccuGuuuuGcuusdTdT 260 PCSK9 (5)(Rp)-uucuAGAccuGuuuuGcusudTdT 261 PCSK9 (6)(Sp)-uucuAGAccuGuuuuGcusudTdT 262 PCSK9 (7)(Rp)-uucuAGAccuGuuuuGcsuudTdT 263 PCSK9 (8)(Sp)-uucuAGAccuGuuuuGcsuudTdT 264 PCSK9 (9)(Rp)-uucuAGAccuGuuuuGscuudTdT 265 PCSK9 (10)(Sp)-uucuAGAccuGuuuuGscuudTdT 266 PCSK9 (11)(Rp)-uucuAGAccuGuuuusGcuudTdT 267 PCSK9 (12)(Sp)-uucuAGAccuGuuuusGcuudTdT 268 PCSK9 (13)(Rp)-uucuAGAccuGuuusuGcuudTdT 269 PCSK9 (14)(Sp)-uucuAGAccuGuuusuGcuudTdT 270 PCSK9 (15)(Rp)-uucuAGAccuGuusuuGcuudTdT 271 PCSK9 (16)(Sp)-uucuAGAccuGuusuuGcuudTdT 272 PCSK9 (17)(Rp)-uucuAGAccuGusuuuGcuudTdT 273 PCSK9 (18)(Sp)-uucuAGAccuGusuuuGcuudTdT 274 PCSK9 (19)(Rp)-uucuAGAccuGsuuuuGcuudTdT 275 PCSK9 (20)(Sp)-uucuAGAccuGsuuuuGcuudTdT 276 PCSK9 (21)(Rp)-uucuAGAccusGuuuuGcuudTdT 277 PCSK9 (22)(Sp)-uucuAGAccusGuuuuGcuudTdT 278 PCSK9 (23)(Rp)-uucuAGAccsuGuuuuGcuudTdT 279 PCSK9 (24)(Sp)-uucuAGAccsuGuuuuGcuudTdT 280 PCSK9 (25)(Rp)-uucuAGAcscuGuuuuGcuudTdT 281 PCSK9 (26)(Sp)-uucuAGAcscuGuuuuGcuudTdT 282 PCSK9 (27)(Rp)-uucuAGAsccuGuuuuGcuudTdT 283 PCSK9 (28)(Sp)-uucuAGAsccuGuuuuGcuudTdT 284 PCSK9 (29)(Rp)-uucuAGsAccuGuuuuGcuudTdT 285 PCSK9 (30)(Sp)-uucuAGsAccuGuuuuGcuudTdT 286 PCSK9 (31)(Rp)-uucuAsGAccuGuuuuGcuudTdT 287 PCSK9 (32)(Sp)-uucuAsGAccuGuuuuGcuudTdT 288 PCSK9 (33)(Rp)-uucusAGAccuGuuuuGcuudTdT 289 PCSK9 (34)(Sp)-uucusAGAccuGuuuuGcuudTdT 290 PCSK9 (35)(Rp)-uucsuAGAccuGuuuuGcuudTdT 291 PCSK9 (36)(Sp)-uucsuAGAccuGuuuuGcuudTdT 292 PCSK9 (37)(Rp)-uuscuAGAccuGuuuuGcuudTdT 293 PCSK9 (38)(Sp)-uuscuAGAccuGuuuuGcuudTdT 294 PCSK9 (38)(Rp)-usucuAGAccuGuuuuGcuudTdT 295 PCSK9 (40)(Sp)-usucuAGAccuGuuuuGcuudTdT 296 NOTE: lower case letters represent2′-OMe RNA residues; capital letters represent RNA residues; d =2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

Synthesis examples for Human PCSK9 siRNA Antisense Strands havingseveral chiral phosphorothioate internucleotide linkages and full chiralphosphorothioate internucleotide linkages.

SEQ Human PCSK9 ID siRNA Antisense Strands NO: PCSK9 (41)(Rp)-AAGcAAAAcAGGUCuAGAAdTsdT 297 PCSK9 (42)(Sp)-AAGcAAAAcAGGUCuAGAAdTsdT 298 PCSK9 (43)(Rp)-AAGcAAAAcAGGUCuAGAAsdTdT 299 PCSK9 (44)(Sp)-AAGcAAAAcAGGUCuAGAAsdTdT 300 PCSK9 (45)(Rp)-AAGcAAAAcAGGUCuAGAsAdTdT 301 PCSK9 (46)(Sp)-AAGcAAAAcAGGUCuAGAsAdTdT 302 PCSK9 (47)(Rp)-AAGcAAAAcAGGUCuAGsAAdTdT 303 PCSK9 (48)(Sp)-AAGcAAAAcAGGUCuAGsAAdTdT 304 PCSK9 (49)(Rp)-AAGcAAAAcAGGUCuAsGAAdTdT 305 PCSK9 (50)(Sp)-AAGcAAAAcAGGUCuAsGAAdTdT 306 PCSK9 (51)(Rp)-AAGcAAAAcAGGUCusAGAAdTdT 307 PCSK9 (52)(Sp)-AAGcAAAAcAGGUCusAGAAdTdT 308 PCSK9 (53)(Rp)-AAGcAAAAcAGGUCsuAGAAdTdT 309 PCSK9 (54)(Sp)-AAGcAAAAcAGGUCsuAGAAdTdT 310 PCSK9 (55)(Rp)-AAGcAAAAcAGGUsCuAGAAdTdT 311 PCSK9 (56)(Sp)-AAGcAAAAcAGGUsCuAGAAdTdT 312 PCSK9 (57)(Rp)-AAGcAAAAcAGGsUCuAGAAdTdT 313 PCSK9 (58)(Sp)-AAGcAAAAcAGGsUCuAGAAdTdT 314 PCSK9 (59)(Rp)-AAGcAAAAcAGsGUCuAGAAdTdT 315 PCSK9 (60)(Sp)-AAGcAAAAcAGsGUCuAGAAdTdT 316 PCSK9 (61)(Rp)-AAGcAAAAcAsGGUCuAGAAdTdT 317 PCSK9 (62)(Sp)-AAGcAAAAcAsGGUCuAGAAdTdT 318 PCSK9 (63)(Rp)-AAGcAAAAcsAGGUCuAGAAdTdT 319 PCSK9 (64)(Sp)-AAGcAAAAcsAGGUCuAGAAdTdT 320 PCSK9 (65)(Rp)-AAGcAAAAscAGGUCuAGAAdTdT 321 PCSK9 (66)(Sp)-AAGcAAAAscAGGUCuAGAAdTdT 322 PCSK9 (67)(Rp)-AAGcAAAsAcAGGUCuAGAAdTdT 323 PCSK9 (68)(Sp)-AAGcAAAsAcAGGUCuAGAAdTdT 324 PCSK9 (69)(Rp)-AAGcAAsAAcAGGUCuAGAAdTdT 325 PCSK9 (70)(Sp)-AAGcAAsAAcAGGUCuAGAAdTdT 326 PCSK9 (71)(Rp)-AAGcAsAAAcAGGUCuAGAAdTdT 327 PCSK9 (72)(Sp)-AAGcAsAAAcAGGUCuAGAAdTdT 328 PCSK9 (73)(Rp)-AAGcsAAAAcAGGUCuAGAAdTdT 329 PCSK9 (74)(Sp)-AAGcsAAAAcAGGUCuAGAAdTdT 330 PCSK9 (75)(Rp)-AAGscAAAAcAGGUCuAGAAdTdT 331 PCSK9 (76)(Sp)-AAGscAAAAcAGGUCuAGAAdTdT 332 PCSK9 (77)(Rp)-AAsGcAAAAcAGGUCuAGAAdTdT 333 PCSK9 (78)(Sp)-AAsGcAAAAcAGGUCuAGAAdTdT 334 PCSK9 (77)(Rp)-AsAGcAAAAcAGGUCuAGAAdTdT 335 PCSK9 (78)(Sp)-AsAGcAAAAcAGGUCuAGAAdTdT 336 PCSK9 (79) (Rp, Sp)- 337AAGcAAAAcAGGUCuAGAAsdTsdT  PCSK9 (80) (Sp, Sp)- 338AAGcAAAAcAGGUCuAGAAsdTsdT PCSK9 (81) (Rp, Sp)- 339AAGcAAAAcAGGUCuAGAsAdTsdT PCSK9 (82) (Sp, Sp)- 340AAGcAAAAcAGGUCuAGAsAdTsdT PCSK9 (83) (Rp, Sp)- 341AAGcAAAAcAGGUCuAGsAAdTsdT PCSK9 (84) (Sp, Sp)- 342AAGcAAAAcAGGUCuAGsAAdTsdT PCSK9 (85) (Rp, Sp)- 343AAGcAAAAcAGGUCuAsGAAdTsdT PCSK9 (86) (Sp, Sp)- 344AAGcAAAAcAGGUCuAsGAAdTsdT PCSK9 (87) (Rp, Sp)- 345AAGcAAAAcAGGUCusAGAAdTsdT PCSK9 (88) (Sp, Sp)- 346AAGcAAAAcAGGUCusAGAAdTsdT PCSK9 (89) (Rp, Sp)- 347AAGcAAAAcAGGUCsuAGAAdTsdT PCSK9 (90) (Sp, Sp)- 348AAGcAAAAcAGGUCsuAGAAdTsdT PCSK9 (91) (Rp, Sp)- 349AAGcAAAAcAGGUsCuAGAAdTsdT PCSK9 (92) (Sp, Sp)- 350AAGcAAAAcAGGUsCuAGAAdTsdT PCSK9 (93) (Rp, Sp)- 351AAGcAAAAcAGGsUCuAGAAdTsdT PCSK9 (94) (Sp, Sp)- 352AAGcAAAAcAGGsUCuAGAAdTsdT PCSK9 (95) (Rp, Sp)- 353AAGcAAAAcAGsGUCuAGAAdTsdT PCSK9 (96) (Sp, Sp)- 354AAGcAAAAcAGsGUCuAGAAdTsdT PCSK9 (97) (Rp, Sp)- 355AAGcAAAAcAsGGUCuAGAAdTsdT PCSK9 (98) (Sp, Sp)- 356AAGcAAAAcAsGGUCuAGAAdTsdT PCSK9 (99) (Rp, Sp)- 357AAGcAAAAcsAGGUCuAGAAdTsdT PCSK9 (100) (Sp, Sp)- 358AAGcAAAAcsAGGUCuAGAAdTsdT PCSK9 (101) (Rp, Sp)- 359AAGcAAAAscAGGUCuAGAAdTsdT PCSK9 (102) (Sp, Sp)- 360AAGcAAAAscAGGUCuAGAAdTsdT PCSK9 (103) (Rp, Sp)- 361AAGcAAAsAcAGGUCuAGAAdTsdT PCSK9 (104) (Sp, Sp)- 362AAGcAAAsAcAGGUCuAGAAdTsdT PCSK9 (105) (Rp, Sp)- 363AAGcAAsAAcAGGUCuAGAAdTsdT PCSK9 (106) (Sp, Sp)- 364AAGcAAsAAcAGGUCuAGAAdTsdT PCSK9 (107) (Rp, Sp)- 365AAGcAsAAAcAGGUCuAGAAdTsdT PCSK9 (108) (Sp, Sp)- 366AAGcAsAAAcAGGUCuAGAAdTsdT PCSK9 (109) (Rp, Sp)- 367AAGcsAAAAcAGGUCuAGAAdTsdT PCSK9 (110) (Sp, Sp)- 368AAGcsAAAAcAGGUCuAGAAdTsdT PCSK9 (111) (Rp, Sp)- 369AAGscAAAAcAGGUCuAGAAdTsdT PCSK9 (112) (Sp, Sp)- 370AAGscAAAAcAGGUCuAGAAdTsdT PCSK9 (113) (Rp, Sp)- 371AAsGcAAAAcAGGUCuAGAAdTsdT PCSK9 (114) (Sp, Sp)- 372AAsGcAAAAcAGGUCuAGAAdTsdT PCSK9 (115) (Rp, Sp)- 373AsAGcAAAAcAGGUCuAGAAdTsdT PCSK9 (116) (Sp, Sp)- 374AsAGcAAAAcAGGUCuAGAAdTsdT PCSK9 (117) (Rp, Sp, Sp, Sp, Rp, 375Sp, Sp, Sp, Rp, Rp)- AsAsGscAsAAsAscsAGGUCuAGAsAsdTsdT PCSK9 (118)(Sp, Rp, Rp, Rp, Sp, 376 Rp, Rp, Rp, Sp, Sp)-AsAsGscAsAAsAscsAGGUCuAGAsAsdTsdT NOTE: lower case letters represent2′-OMe RNA residues; capital letters represent RNA residues; d =2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

EXAMPLE 3 Stereopure FOXO-1 antisense analogs.

Rational Design—Chirally Controlled Antisense OligonucleotideCompositions

The unprecedented nuclease stability determined in vivo and in a wholerat liver homogenate model of the Sp-chiral phosphorothioateinternucleotide linkage is applied in the novel design of new types ofRNaseH substrate gapmers, whereby the external flanks are composed ofunmodified DNA and the internal gap core is modified with 2′ chemicalmodifications (2′OMe, 2′MOE, 2′LNA, 2′F, etc). Eventually this design isextended to fully unmodified DNA therapeutic oligonucleotides whereincareful chiral control of the phosphorothioate backbone confers thedesired pharmacological properties of the RNaseH therapeuticoligonucleotide.

The application of the triplet-phosphate repeating motif designed afterstudying the crystal structure of human RNaseH has been employed aswell. The crystal structure of RNaseH has been previously published(Structure of Human RNase H1 Complexed with an RNA/DNA Hybrid: Insightinto HIV Reverse Transcription, Nowotny et al., Molecular Cell, Volume28, Issue 2, 264-276, 2007, pdb file: 2qkb). Among other things, thepresent invention recognizes the importance of internucleotidic linkagestereochemistry of oligonucleotides, for example, in settings herein.Upon performing in silico analysis upon this structure using the programPymol, Applicant found that the phosphate-binding pocket of Human RNaseH1 makes polar contacts with three contiguous phosphates of thecomplexed DNA, and interacts preferentially with the Pro-R/Pro-R/Pro-S(or with the Pro-S/Pro-S/Pro-R) respective oxygen atoms of each of thesethree phosphates. Based on this observation we designed two chiralarchitectures with repeating (RRS) and (SSR) triplet phosphorothioatesmotifs as designed RNase H substrates. Applicant also designed otherinternucleotidic linkage stereochemical patterns. As demonstrated byexemplary results provide herein, provided chirally controlledoligonucleotide compositions of oligonucleotide types that comprisescertain backbone internucleotidic linkage patterns (patterns backbonechiral centers) provides significantly increased activity and/orkinetics. Among others, a sequence of 5′-RSS-3′ backbone chiral centersis particularly useful and delivers unexpected results as described inthe present disclosure.

The combination of increased Sp chiral backbone (for enzymatic stabilityand other pharmacologically advantageous properties) and (RRS) or (SSR)repeating triplet chiral backbone motifs (for enhancing the property asRNase H substrate) are also utilized in the novel designs.; “S”represents Sp-phosphorothioate linkage and “R” representsRp-phosphorothioate linkage.

Another alternative design is based on the increased amount of Sp chiralphosphorothioate backbone in extended repeating motifs such as: (SSSR)n,SR(SSSR)n, SSR(SSSR)n, SSR(SSSR)n; (SSSSR)n, SR(SSSSR)n, SSR(SSSSR)n,SSR(SSSSR)n, SSSR(SSSSR)n; (SSSSSR)n; SR(SSSSSR)n, SSR(SSSSSR)n,SSR(SSSSSR)n, SSSR(SSSSSR)n, SSSSR(SSSSSR)n; etc., where n=0-50depending on the number of respective internucleotide linkages.; “S”represents Sp-phosphorothioate linkage and “R” representsRp-phosphorothioate linkage. In some embodiments, n is 0. In someembodiments, R is 1-50. In some embodiments, R is 1. In someembodiments, a common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises a motifdescribed herein. In some embodiments, a motif is in the core region. Insome embodiments, n is 0. In some embodiments, R is 1-50. In someembodiments, R is 1. In some embodiments, n is 2. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 5.

Another alternative design is based on the “invert” architecture designof the stereo backbone (“stereo invert-mers”). These result frompositioning the stereochemistry of the chiral phosphorothioate in ainverting manner, exposing some Sp-rich motifs at the 5′ and 3′ endextremities of the oligonucleotide as well as the middle portion of theoligonucleotide and having the repeating stereochemistry motifspositioned in a invert image manner on both sides, such as:

SS(SSR)n(SSS)(RSS)nSS;

SS(SSR)n(SRS)(RSS)nSS;

SS(SSR)n(SSR)(RSS)nSS;

SS(SSR)n(RSS)(RSS)nSS;

SS(RSS)n(SSS)(SSR)nSS;

SS(RSS)n(SRS)(SSR)nSS;

SS(RSS)n(SSR)(SSR)nSS;

SS(RSS)n(RSS)(SSR)nSS; etc.,

where n=0-50, depending on the number of respective internucleotidelinkages. ; “S” represents Sp-phosphorothioate linkage and “R”represents Rp-phosphorothioate linkage. In some embodiments, a commonpattern of backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises a motif described herein. In someembodiments, a motif is in the core region. In some embodiments, n is 0.In some embodiments, n is 1. In some embodiments, n is 1-50. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5.Initial ScreenSynthesis: Summary for Oligonucleotide Synthesis on a DNA/RNASynthesizer MerMade-12 (2′-deoxy and 2′—OMe cycle)

delivery volume wait time step reaction reagent (mL) (sec) 1detritylation 3% TCA in DCM 4 × 1 N. A. 2 coupling 0.15M 2 × 0.5 mL 60 + 60 (DNA), phosphoramidite in 300 + 300 ACN + 0.45M ETT (2′-OMeRNA)in ACN 3 capping 5% Pac₂O in THF/ 1 60 2,6-lutidine + 16% NMI in THF 4oxidation 0.02 Iodine in 1 240 water/pyridine

Stereorandom PS oligonucleotides having DNA-2′-OMe-DNA (7-6-7) design:ONT-141 d(CsCsCsTsCsTsGs)gsaststsgsasd(GsCsAsTsCsCsA) (SEQ ID NO: 377)ONT-142 d(AsAsGsCsTsTsTs)gsgststsgsgsd(GsCsAsAsCsAsC) (SEQ ID NO: 378)ONT-143 d(AsGsTsCsAsCsTs)tsgsgsgsasgsd(CsTsTsCsTsCsC) (SEQ ID NO: 379)ONT-144 d(CsAsCsTsTsGsGs)gsasgscststsd(CsTsCsCsTsGsG) (SEQ ID NO: 380)ONT-145 d(AsTsAsGsCsCsAs)tstsgscsasgsd(CsTsGsCsTsCsA) (SEQ ID NO: 381)ONT-146 d(TsGsGsAsTsTsGs)asgscsastscsd(CsAsCsCsAsAsG) (SEQ ID NO: 382)ONT-147 d(CsCsAsTsAsGsCs)csaststsgscsd(AsGsCsTsGsCsT) (SEQ ID NO: 383)ONT-148 d(GsTsCsAsCsTsTs)gsgsgsasgscsd(TsTsCsTsCsCsT) (SEQ ID NO: 384)ONT-149 d(CsCsAsGsGsGsCs)ascstscsastsd(CsTsGsCsAsTsG) (SEQ ID NO: 385)ONT-150 d(GsCsCsAsTsCsCs)asasgstscsasd(CsTsTsGsGsGsA) (SEQ ID NO: 386)ONT-151 d(GsAsAsGsCsTsTs)tsgsgststsgsd(GsGsCsAsAsCsA) (SEQ ID NO: 387)ONT-152 d(CsTsGsGsAsTsTs)gsasgscsastsd(CsCsAsCsCsAsA) (SEQ ID NO: 388)ONT-183 d(CsAsAsGsTsCsAs)cststsgsgsgsd(AsGsCsTsTsCsT) (SEQ ID NO: 389)ONT-184 d(AsTsGsCsCsAsTs)cscsasasgstsd(CsAsCsTsTsGsG) (SEQ ID NO: 390)ONT-185 d(AsTsGsAsGsAsTs)gscscstsgsgsd(CsTsGsCsCsAsT) (SEQ ID NO: 391)ONT-186 d(TsTsGsGsGsAsGs)cststscstscsd(CsTsGsGsTsGsG) (SEQ ID NO: 392)ONT-187 d(TsGsGsGsAsGsCs)tstscstscscsd(TsGsGsTsGsGsA) (SEQ ID NO: 393)ONT-188 d(TsTsAsTsGsAsGs)astsgscscstsd(GsGsCsTsGsCsC) (SEQ ID NO: 394)ONT-189 d(GsTsTsAsTsGsAs)gsastsgscscsd(TsGsGsCsTsGsC) (SEQ ID NO: 395)ONT-190 d(CsCsAsAsGsTsCs)ascststsgsgsd(GsAsGsCsTsTsC) (SEQ ID NO: 396)ONT-191 d(AsGsCsTsTsTsGs)gststsgsgsgsd(CsAsAsCsAsCsA) (SEQ ID NO: 397)ONT-192 d(TsAsTsGsAsGsAs)tsgscscstsgsd(GsCsTsGsCsCsA) (SEQ ID NO: 398)ONT-193 d(TsGsTsTsAsTsGs)asgsastsgscsd(CsTsGsGsCsTsG) (SEQ ID NO: 399)ONT-194 d(AsTsCsCsAsAsGs)tscsascststsd(GsGsGsAsGsCsT) (SEQ ID NO: 400)ONT-195 d(GsGsGsAsAsGsCs)tststsgsgstsd(TsGsGsGsCsAsA) (SEQ ID NO: 401)ONT-196 d(CsTsCsCsAsTsCs)csastsgsasgsd(GsTsCsAsTsTsC) (SEQ ID NO: 402)ONT-197 d(AsAsGsTsCsAsCs)tstsgsgsgsasd(GsCsTsTsCsTsC) (SEQ ID NO: 403)ONT-198 d(CsCsAsTsCsCsAs)asgstscsascsd(TsTsGsGsGsAsG) (SEQ ID NO: 404)ONT-199 d(TsCsCsAsAsGsTs)csascststsgsd(GsGsAsGsCsTsT) (SEQ ID NO: 405)ONT-200 d(CsCsTsCsTsGsGs)aststsgsasgsd(CsAsTsCsCsAsC) (SEQ ID NO: 406)ONT-201 d(AsCsTsTsGsGsGs)asgscststscsd(TsCsCsTsGsGsT) (SEQ ID NO: 407)ONT-202 d(CsTsTsGsGsGsAs)gscststscstsd(CsCsTsGsGsTsG) (SEQ ID NO: 408)ONT-203 d(CsAsTsGsCsCsAs)tscscsasasgsd(TsCsAsCsTsTsG) (SEQ ID NO: 409)ONT-204 d(TsGsCsCsAsTsCs)csasasgstscsd(AsCsTsTsGsGsG) (SEQ ID NO: 410)ONT-205 d(TsCsCsAsTsCsCs)astsgsasgsgsd(TsCsAsTsTsCsC) (SEQ ID NO: 411)ONT-206 d(AsGsGsGsCsAsCs)tscsastscstsd(GsCsAsTsGsGsG) (SEQ ID NO: 412)ONT-207 d(CsCsAsGsTsTsCs)cststscsastsd(TsCsTsGsCsAsC) (SEQ ID NO: 413)ONT-208 d(CsAsTsAsGsCsCs)aststsgscsasd(GsCsTsGsCsTsC) (SEQ ID NO: 414)ONT-209 d(TsCsTsGsGsAsTs)tsgsasgscsasd(TsCsCsAsCsCsA) (SEQ ID NO: 415)ONT-210 d(GsGsAsTsTsGsAs)gscsastscscsd(AsCsCsAsAsGsA) (SEQ ID NO: 416)Biology in vitro Data in HepG2 Cells for the initial DNA-2′-OMe-DNA(7-6-7) design: (d Upper case)=DNA; lower case=2′-OMe;s=phosphorothioate.FOXO1

Levels at 20 nM (%) SD ONT-141 89 6 ONT-142 45 1 ONT-143 98 2 ONT-144 891 ONT-145 46 5 ONT-146 99 1 ONT-147 66 6 ONT-148 101 2 ONT-149 95 6ONT-150 58 4 ONT-151 41 5 ONT-152 84 5 ONT-183 95 2 ONT-184 58 4 ONT-18542 2 ONT-186 96 4 ONT-187 92 3 ONT-188 47 5 ONT-189 63 5 ONT-190 83 2ONT-191 58 4 ONT-192 46 2 ONT-193 58 2 ONT-194 76 1 ONT-195 66 0 ONT-19677 2 ONT-197 90 6 ONT-198 42 4 ONT-199 68 1 ONT-200 89 6 ONT-201 91 2ONT-202 94 2 ONT-203 86 1 ONT-204 58 2 ONT-205 75 3 ONT-206 94 5 ONT-20796 0 ONT-208 54 0 ONT-209 87 4 ONT-210 92 4

FOXO1

Levels at 200 nM (%) SD ONT-141 37 4 ONT-142 45 4 ONT-143 46 2 ONT-14442 5 ONT-145 53 4 ONT-146 31 2 ONT-147 28 8 ONT-148 45 4 ONT-149 29 5ONT-150 32 6 ONT-151 38 4 ONT-152 30 5 ONT-183 60 5 ONT-184 34 2 ONT-18550 2 ONT-186 86 3 ONT-187 76 6 ONT-188 50 5 ONT-189 38 2 ONT-190 51 1ONT-191 43 5 ONT-192 54 7 ONT-193 41 6 ONT-194 50 1 ONT-195 43 6 ONT-19633 7 ONT-197 57 4 ONT-198 40 5 ONT-199 50 5 ONT-200 28 9 ONT-201 46 6ONT-202 57 9 ONT-203 27 7 ONT-204 36 6 ONT-205 29 5 ONT-206 81 0 ONT-20737 4 ONT-208 43 3 ONT-209 35 4 ONT-210 40 4

Stereorandom PS oligonucleotides having 2′-OMe-DNA-2′OMe (3-14-3) design:(d Upper case) = DNA; lower case = 2′-OMe; s = phosphorothioate. ONT-129cscscsd(TsCsTsGsGsAsTsTsGsAsGsCsAsTs)cscsa (SEQ ID NO: 417) ONT-130asasgsd(CsTsTsTsGsGsTsTsGsGsGsCsAsAs)csasc (SEQ ID NO: 418) ONT-131asgstsd(CsAsCsTsTsGsGsGsAsGsCsTsTsCs)tscsc (SEQ ID NO: 419) ONT-132csascsd(TsTsGsGsGsAsGsCsTsTsCsTsCsCs)tsgsg (SEQ ID NO: 420) ONT-133astsasd(GsCsCsAsTsTsGsCsAsGsCsTsGsCs)tscsa (SEQ ID NO: 421) ONT-134tsgsgsd(AsTsTsGsAsGsCsAsTsCsCsAsCsCs)asasg (SEQ ID NO: 422) ONT-135cscsasd(TsAsGsCsCsAsTsTsGsCsAsGsCsTs)gscst (SEQ ID NO: 423) ONT-136gstscsd(AsCsTsTsGsGsGsAsGsCsTsTsCsTs)cscst (SEQ ID NO: 424) ONT-137cscsasd(GsGsGsCsAsCsTsCsAsTsCsTsGsCs)astsg (SEQ ID NO: 425) ONT-138gscscsd(AsTsCsCsAsAsGsTsCsAsCsTsTsGs)gsgsa (SEQ ID NO: 426) ONT-139gsasasd(GsCsTsTsTsGsGsTsTsGsGsGsCsAs)ascsa (SEQ ID NO: 427) ONT-140cstsgsd(GsAsTsTsGsAsGsCsAsTsCsCsAsCs)csasa (SEQ ID NO: 428) ONT-155csasasd(GsTsCsAsCsTsTsGsGsGsAsGsCsTs)tscst (SEQ ID NO: 429) ONT-156astsgsd(CsCsAsTsCsCsAsAsGsTsCsAsCsTs)tsgsg (SEQ ID NO: 430) ONT-157astsgsd(AsGsAsTsGsCsCsTsGsGsCsTsGsCs)csast (SEQ ID NO: 431) ONT-158tstsgsd(GsGsAsGsCsTsTsCsTsCsCsTsGsGs)tsgsg (SEQ ID NO: 432) ONT-159tsgsgsd(GsAsGsCsTsTsCsTsCsCsTsGsGsTs)gsgsa (SEQ ID NO: 433) ONT-160tstsasd(TsGsAsGsAsTsGsCsCsTsGsGsCsTs)gscsc (SEQ ID NO: 434) ONT-161gststsd(AsTsGsAsGsAsTsGsCsCsTsGsGsCs)tsgsc (SEQ ID NO: 435) ONT-162cscsasd(AsGsTsCsAsCsTsTsGsGsGsAsGsCs)tstsc (SEQ ID NO: 436) ONT-163asgscsd(TsTsTsGsGsTsTsGsGsGsCsAsAsCs)ascsa (SEQ ID NO: 437) ONT-164tsastsd(GsAsGsAsTsGsCsCsTsGsGsCsTsGs)cscsa (SEQ ID NO: 438) ONT-165tsgstsd(TsAsTsGsAsGsAsTsGsCsCsTsGsGs)cstsg (SEQ ID NO: 439) ONT-166astscsd(CsAsAsGsTsCsAsCsTsTsGsGsGsAs)gscst (SEQ ID NO: 440) ONT-167gsgsgsd(AsAsGsCsTsTsTsGsGsTsTsGsGsGs)csasa (SEQ ID NO: 441) ONT-168cstscsd(CsAsTsCsCsAsTsGsAsGsGsTsCsAs)tstsc (SEQ ID NO: 442) ONT-169asasgsd(TsCsAsCsTsTsGsGsGsAsGsCsTsTs)cstsc (SEQ ID NO: 443) ONT-170cscsasd(TsCsCsAsAsGsTsCsAsCsTsTsGsGs)gsasg (SEQ ID NO: 444) ONT-171tscscsd(AsAsGsTsCsAsCsTsTsGsGsGsAsGs)cstst (SEQ ID NO: 445) ONT-172cscstsd(CsTsGsGsAsTsTsGsAsGsCsAsTsCs)csasc (SEQ ID NO: 446) ONT-173ascstsd(TsGsGsGsAsGsCsTsTsCsTsCsCsTs)gsgst (SEQ ID NO: 447) ONT-174cststsd(GsGsGsAsGsCsTsTsCsTsCsCsTsGs)gstsg (SEQ ID NO: 448) ONT-175csastsd(GsCsCsAsTsCsCsAsAsGsTsCsAsCs)tstsg (SEQ ID NO: 449) ONT-176tsgscsd(CsAsTsCsCsAsAsGsTsCsAsCsTsTs)gsgsg (SEQ ID NO: 450) ONT-177tscscsd(AsTsCsCsAsTsGsAsGsGsTsCsAsTs)tscsc (SEQ ID NO: 451) ONT-178asgsgsd(GsCsAsCsTsCsAsTsCsTsGsCsAsTs)gsgsg (SEQ ID NO: 452) ONT-179cscsasd(GsTsTsCsCsTsTsCsAsTsTsCsTsGs)csasc (SEQ ID NO: 453) ONT-180csastsd(AsGsCsCsAsTsTsGsCsAsGsCsTsGs)cstsc (SEQ ID NO: 454) ONT-181tscstsd(GsGsAsTsTsGsAsGsCsAsTsCsCsAs)cscsa (SEQ ID NO: 455) ONT-182gsgsasd(TsTsGsAsGsCsAsTsCsCsAsCsCsAs)asgsa (SEQ ID NO: 456)Biology In Vitro Data in HepG2 cells for the 2′-OMe-DNA-2′-OMe (3-14-3)Design:FOXO1

Levels at 20 nM (%) SD ONT-129 82 5 ONT-130 49 4 ONT-131 92 3 ONT-132 912 ONT-133 58 3 ONT-134 73 2 ONT-135 65 5 ONT-136 92 2 ONT-137 94 2ONT-138 78 1 ONT-139 61 1 ONT-140 82 4 ONT-155 95 2 ONT-156 74 1 ONT-15756 2 ONT-158 93 1 ONT-159 94 1 ONT-160 71 1 ONT-161 67 1 ONT-162 89 1ONT-163 55 7 ONT-164 68 4 ONT-165 70 1 ONT-166 89 4 ONT-167 81 0 ONT-16881 0 ONT-169 94 0 ONT-170 88 1 ONT-171 92 4 ONT-172 86 2 ONT-173 90 1ONT-174 93 2 ONT-175 84 1 ONT-176 80 2 ONT-177 83 2 ONT-178 95 2 ONT-17993 8 ONT-180 68 7 ONT-181 85 5 ONT-182 80 5

FOXO1

Levels at 200 nM (%) SD ONT-129 27 1 ONT-130 46 4 ONT-131 53 9 ONT-13253 2 ONT-133 48 6 ONT-134 35 9 ONT-135 45 15 ONT-136 40 7 ONT-137 50 4ONT-138 80 3 ONT-139 40 3 ONT-140 33 13 ONT-155 52 2 ONT-156 35 4ONT-157 39 2 ONT-158 87 6 ONT-159 89 5 ONT-160 33 10 ONT-161 40 11ONT-162 60 7 ONT-163 42 8 ONT-164 34 10 ONT-165 38 1 ONT-166 62 9ONT-167 64 1 ONT-168 38 2 ONT-169 67 3 ONT-170 74 8 ONT-171 65 5 ONT-17233 18 ONT-173 72 15 ONT-174 65 15 ONT-175 38 21 ONT-176 48 8 ONT-177 285 ONT-178 97 11 ONT-179 47 6 ONT-180 56 12 ONT-181 45 26 ONT-182 33 17

Hit selection: ONT-151 d(GsAsAsGsCsTsTs)tsgsgststsgsd(GsGsCsAsAsCsA)(SEQ ID NO: 457) ONT-198 d(CsCsAsTsCsCsAs)asgstscsascsd(TsTsGsGsGsAsG)(SEQ ID NO: 458) ONT-185 d(AsTsGsAsGsAsTs)gscscstsgsgsd(CsTsGsCsCsAsT)(SEQ ID NO: 459) ONT-142 d(AsAsGsCsTsTsTs)gsgststsgsgsd(GsCsAsAsCsAsC)(SEQ ID NO: 460) ONT-145 d(AsTsAsGsCsCsAs)tstsgscsasgsd(CsTsGsCsTsCsA)(SEQ ID NO: 461) ONT-192 d(TsAsTsGsAsGsAs)tsgscscstsgsd(GsCsTsGsCsCsA)(SEQ ID NO: 462) ONT-188 d(TsTsAsTsGsAsGs)astsgscscstsd(GsGsCsTsGsCsC)(SEQ ID NO: 463)

step reaction reagent delivery vol (mL) wait time (sec) 1 detritylation3% TCA in DCM 4 × 1   N. A. 2 coupling 0.15 M chiral 2 × 0.5 2 × 450(2′-OMe phosphoramidite in RNA) ACN + 2 M CMPT in 2 × 300 (DNA) ACN 3capping 1 5% Pac₂O in THF/2,6- 1 60 lutidine 4 capping 2 5% Pac₂O inTHF/2,6- 1 60 lutidine + 16% NMI in THF 5 sulfurization 0.3 MS-(2-cyanoethyl) methylthiosulfonate  

  in ACN/BSTFA 1 600Examples Applied on the FOXO1 Hit Sequences:

Examples include but are not limited to:

(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 464)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 465)

(Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,Sp)d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 466)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 467)

(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 468)

(Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp,Sp) d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 469)

(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 470)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 471)

(Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 472)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 473)

(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp,Sp) d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 474)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAs](AsGsTsCsAsCs) _(OMe)d[TsTsGsGsGsAsG] (SEQ ID NO:475)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTs](GsCsCsTsGsGs) _(OMe) d[CsTsGsCsCsAsT] (SEQ ID NO:476)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAs](AsGsTsCsAsCs) _(LNA)d[TsTsGsGsGsAsG] (SEQ ID NO:477)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTs](GsCsCsTsGsGs) _(LNA) d[CsTsGsCsCsAsT] (SEQ ID NO:478)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAs](AsGsTsCsAsCs) _(MOE)d[TsTsGsGsGsAsG] (SEQ ID NO:479)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTs](GsCsCsTsGsGs) _(MOE) d[CsTsGsCsCsAsT] (SEQ ID NO:480)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp,Sp)(CsCsAs)_(OMe)d[TsCsCsAsAsGsTsCsAsCsTsTsGsGs](GsAsG)_(OMe) (SEQ IDNO: 481)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp,Sp)(AsTsGs)_(MOE)d[AsGsAsTsGsCsCsTsGsGsCsTsGsCs](CsAsT)_(MOE) (SEQ IDNO: 482)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)(CsCsAs)_(LNA)d[TsCsCsAsAsGsTsCsAsCsTsTsGsGs](GsAsG)_(LNA) (SEQ IDNO: 483)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)(AsTsGs)_(OMe)d[AsGsAsTsGsCsCsTsGsGsCsTsGsCs](CsAsT)_(OMe) (SEQ IDNO: 484)

(Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 485)

(Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 486)

(Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp,Sp) d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 487)

(Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 488)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 489)

(Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 490)

(Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp,Sp) d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 491)

(Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 492)

(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 493)

(Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 494)

(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 495)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 496)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 497)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 498)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 499)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 500)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 501)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)(CsCs)_(OMe)d[AsTsCsCsAsAs](GsTsCs)_(OMe)d[AsCsTsTsGsGsGs](AsG)_(OMe)(SEQ ID NO: 502)

(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp) (CsCs)_(LNA)d[AsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGs](AsG)_(LNA) (SEQ IDNO: 503)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp) (CsCs)_(MOE)d[AsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGs](AsG)_(MOE) (SEQ IDNO: 504)

(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp) (CsCs)_(OMe)d[AsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGs](AsG)_(OMe) (SEQ IDNO: 505)

EXAMPLE 4 Suppression of Nucleic Acid Polymer

Among other things, the present invention provides chirally controlledoligonucleotide compositions and methods thereof that deliver unexpectedresults when, e.g., used for suppressing nucleic acid polymers through,in some cases, cleavage of such nucleic acid polymers. Examples includebut are not limited to those presented herein.

RNase H Assay

Cleavage rate of nucleic acid polymers by nucleases, for example, RNA byRNase H, is important with respect to the use of oligonucleotides intherapeutic technologies such as antisense technology. Using our assay,we investigated the cleavage rates and analyzed the metabolites forchirally controlled oligonucleotide compositions of particularoligonucleotide types (P-diastereomers) when oligonucleotides of theparticular oligonucleotide types are bound to complementary RNA. Resultsbelow also illustrate the importance of cleavage patterns recognized bythe present invention.

RNase H used herein is a ubiquitously expressed endonuclease thathydrolyses the RNA strand of a RNA/DNA hybrid. It plays an importantrole in the mode of action of antisense oligonucleotides. In someembodiments, RNase H cleavage rate is significantly reduced when the RNAsubstrate is structured (Lima, W. F., Venkatraman, M., Crooke, S. T. TheInfluence of Antisense Oligonucleotide-induced RNA Structure onEscherichia coli RNase H1 Activity The Journal Of Biological Chemistry272, No. 29, 18191-18199, (1997)). Furthermore, the 2′ —MOE gapmerdesigns (5-10-5) offer higher affinities for RNA targets leading tominimal turnover of the antisense strand. Presence of 2′—MOEmodifications in the wings also reduce the number of RNase H cleavagesites.

To study the RNA cleavage rate, the present invention provides a simpleassay to quantify the length of RNA remaining after incubation withRNase H. The provided method, among other things, provides the relativerates of RNase H cleavage for stereorandom 2′-modified gapmers,stereorandom DNA oligonucleotide compositions and chirally pureP-diastereomers (chirally controlled oligonucleotide compositions of acorresponding oligonucleotide type) for various oligomers for differenttargets. Changing the stereochemistry at 2′-modified regions and the DNAcore provides information with respect to how stereochemistry in theseregions affects the interaction of RNase H to its substrates. RNase Hreaction mixtures at different time points were analyzed by LCMS todetermine the cleavage pattern. The present invention, among otherthings, provides nucleic acid polymer, for example RNA, cleavage ratesand cleavage patterns (maps) that are critical to design stereochemicalnucleic acid architectures for optimal activity, e.g., antisenseactivity.

Equipment:

Alliance HPLC, 2489—TUV, 2695E—Equipped with autosampler

Cary100 (Agilent Technologies)

Methods:

DNA/RNA Duplex Preparation: Oligonucleotide concentrations weredetermined by measuring the absorbance in water at 260 nm. DNA/RNAduplexes were prepared by mixing equimolar solutions oligonucleotideswith each strand concentration of 10 uM. The mixtures were heated at 90°C. for 2 minutes in water bath and were cooled down slowly over severalhours.

Human RNase H Protein Expression and Purification: Human RNase HC clonewas obtained from Prof. Wei Yang's laboratory at NIH Bethesda. Theprotocol for obtaining this human RNase HC (residues 136-286) has beendescribed (Nowotny, M. et al. Structure of Human RNase H1 Complexed withan RNA/DNA Hybrid: Insight into HIV Reverse Transcription. MolecularCell 28, 264-276, (2007). The protein expression was carried out byfollowing reported protocol with the exception that the resultingprotein had an N-terminal His6 tag (SEQ ID NO: 621). BL21(DE3) E.colicells in LB medium were used for protein expression. Cells were grown at37° C. till OD600 reached around 0.7. The cultures were then cooled and0.4 mM IPTG was added to induce protein expression overnight at 16° C.E.coli extract was prepared by sonication in buffer A (40 mM NaH₂PO₄ (pH7.0), 1 M NaCl, 5% glycerol, 2.8 mM β-mercaptoethanol and 10 mMimidazole) with the addition of protease inhibitors (Sigma-Aldrich). Theextract was purified by Ni affinity column using buffer A plus 60 mMimidazole. The protein was eluted with a linear gradient of 60 to 300 mMimidazole. The protein peak was collected and was further purified on aMono S column (GE Healthcare) with a 100 mM -500 mM gradient of NaCl inbuffer B. Fractions containing RNase HC were concentrated to 0.3 mg/mlin the storage buffer (20 mM HEPES (pH 7.0), 100 mM NaCl, 5% glycerol,0.5 mM EDTA, 2 mM DTT) and stored at −20° C. 0.3 mg/ml enzymeconcentration corresponds to 17.4 uM based on its reported extinctioncoefficient (32095 cm⁻¹M⁻¹) and MW (18963.3 Da units).

RNase H assay: In a 96-well plate, to 25 μL DNA/RNA duplex (10 μM) wasadded 5 μL of 10X RNase H buffer followed by 15 μL water. The mixturewas incubated at 37° C. for a few minutes and then 5 μL of 0.1 μM stocksolution of enzyme was added to give total volume of 50 μL with finalsubstrate/enzyme concentration 5 μM/0.01 μM (500:1) and was furtherincubated at 37° C. Various ratios of the DNA/RNA duplex: RNase Hprotein were studied using these conditions to find an optimal ratio tostudy the kinetics. The reactions were quenched at different time pointsusing 10 μL of 500 mM EDTA disodium solution in water. For zero min timepoint, EDTA was added to the reaction mixture before the addition ofenzyme. Controls were run to ensure that EDTA was able to successfullyinhibit the enzyme activity completely. After all the reactions werequenched 10 μL of each reaction mixture was injected on to analyticalHPLC column (XBridge C18, 3.5 um, 4.6×150 mm, Waters Part# 186003034).Kcat/Km can be measured by a number of methods, such as FRET(Fluorescence Resonance Energy Transfer) dependent RNase H assay usingdual labeled RNA and monitored by SpectraMax.

Solid Phase Extraction Protocol for Sample Preparation for LCMS: 96 wellplate (Waters part# 186002321) was used to clean the RNase H reactionmixture before running LCMS. 500 μL of acetonitrile followed by waterwas used to equilibrate the plate under mild vacuum with the help ofmanifold (Millipore part# MSV MHTS00). Precaution was taken not to letthe plate dry. About 50-100 μL of RNase H reaction mixture was loaded ineach well followed by water washings (2 mL) under mild vacuum. 2×500 μLof 70% ACN/Water was used to recover the sample. The recovered sampleswere transferred to 2 mL centrifuge tubes and were concentrated todryness in speed vac. Each dry sample was reconstituted in 100 μL waterand 10 μL was injected on Acquity UPLC@OST C18 1.7 um, 2.1×50 mm (part#186003949) for LCMS analysis.

For mass spectrometry analysis, the reaction mixtures after quenchingwere cleaned using C₁₈ 96 well plate (Waters). The oligomers were elutedin 70% Acetonitrile/Water. The Acetonitrile was evaporated usingspeedvac and the resulting residue was reconstituted in water forinjection.

Eluent A=50 mM Triethyl ammonium acetate

Eluent B=Acetonitrile

Column Temperature=60° C.

UV was recorded at 254 nm and 280 nm

RP-HPLC Gradient Method

Time Flow (min) (ml/min) % A % B Curve 1 0.0 1.00 95.0 5.0 2 2.00 1.0095.0 5.0 1 3 22.00 1.00 80.0 20.0 6 4 25.00 1.00 5.0 95.0 6 5 25.5 1.0095.0 5.0 1 6 30 1.00 95.0 5.0 1

On HPLC chromatograms, peak areas corresponding to full length RNAoligomer (ONT-28) were integrated, normalized using the DNA peak andwere plotted against time (FIG. 8). ONT-87 demonstrated superiorcleavage for complementary RNA when in duplex form, in comparison to theother product candidates and Mipomersen. Since all the diastereomers inthis panel have 2′-MOE modified wings that do not activate RNase Henzyme, without the intention to be limited by theory, Applicant notesthat activity is likely dictated by the stereochemistry in the DNA core.Heteroduplexes with ONT-77 to ONT-81 including Mipomersen in theantisense strand show very similar RNA cleavage rates. ONT-89 withalternating Sp/Rp stereochemistry showed the least activity in thetested time frame under the tested conditions. Among the testedoligonucleotides with MOE modifications, ONT-87 and ONT-88 units in theantisense strand exhibited increased in activity in comparison to therest of the heteroduplexes. Particularly, ONT-87 provided surprisinglyhigh cleavage rate and unexpected low level of remaining target RNA.Additional exemplary data were illustrated in FIG. 6 and FIG. 24.

In Vitro Oligonucleotide Transfection Assay: Transfection assays arewidely known and practiced by persons having ordinary skill in the art.An exemplary protocol is described herein. Hep3B cells are reversetransfected with Lipofectamine 2000 (Life Technologies, Cat. No.11668-019) at 18×10³ cells/well density in 96-well plates using themanufacturer's protocol. For dose response curves eight 1/3 serialdilutions are used starting from 60-100 nM. 25 μL of 6× oligonucleotideconcentration is mixed with a prepared mixture of 0.4 μL Lipofectamine2000 with 25 μL of serum-free medium Opti-MEM medium (Gibco, Cat. No.31985-062) per well. After a 20 min minute incubation, 100 μL of 180×10³cells/ml suspended in 10% FBS in DMEM cell culture media (Gibco, Cat.No. 11965-092) is added to bring the final volume to 150 μL per well.24-48 hours post transfection Hep3B cells are lysed by adding 75 μL ofLysis Mixture with 0.5 mg/ml Proteinase K using QuantiGene SampleProcessing Kit for Cultured Cells (Affymetrix, Cat. No. QS0103). TheTarget mRNA and GAPDH mRNA expression levels in cell lysates aremeasured using Affymetrix QuantiGene 2.0 Assay Kit (Cat. No. QS0011)according to the manufacturer's protocol. The Target mRNA expression isnormalized to GAPDH mRNA expression from the same sample; and relativeTarget/GAPDH levels are compared to transfections using Lipofectamine2000 only (no oligonucleotide) control. Dose response curves aregenerated by GraphPad Prism 6 using nonlinear regression log (inhibitor)vs. response curve fit with variable slope (4 parameters). For exemplaryresults, see FIG. 24, FIG. 27 and FIG. 29.

EXAMPLE 5 Provided Compositions and Methods Provide Control of CleavagePatterns

The present invention surprisingly found that internucleotidic linkagestereochemistry pattern has unexpected impact on cleavage patterns ofnucleic acid polymers. By changing common patterns of backbone chiralcenters of chirally controlled oligonucleotide compositions, numbers ofcleavage sites, cleavage percentage at a cleavage site, and/or locationsof cleavage sits can be surprisingly altered, both independently and incombination. As described in the example herein, provided compositionsand methods can provide control over cleavage patterns of nucleic acidpolymers.

Using similar assay conditions, various chirally controlledoligonucleotide compositions of different oligonucleotide types weretested. Exemplary cleavage patterns of the target RNA sequence ispresented in FIG. 9. Certain pattern of backbone chiral centers, such asthat in ONT-87 and ONT-154, surprisingly produces only one cleavage sitein the target sequence. Moreover, it is surprisingly found thatoligonucleotides providing single cleavage site, such as ONT-87 andONT-154, provide unexpectedly high cleavage rate and low level ofremaining target nucleic acid polymer. See also FIG. 8, FIG. 10 and FIG.11.

EXAMPLE 6 Exemplary Cleavage of FOXO1 mRNA

Oligonucleotide compositions targeting different regions of FOXO1 mRNAwere tested in cleavage assays as described above. In each case,chirally controlled oligonucleotide compositions were shown to becapable of providing altered cleavage patterns relative to referencecleavage patterns from chirally uncontrolled oligonucleotidecompositions sharing the same common base sequence and length. Forexemplary results, see FIG. 10 and FIG. 11. As shown in FIG. 12,exemplary chirally controlled oligonucleotide compositions provide bothsignificantly faster cleavage rates and unexpectedly low levels ofremaining substrates when compared to reference chirally uncontrolledoligonucleotide compositions. In some embodiments, as shown in FIG. 11,the cleavage sites are associated with RpSpSp backbone chiral centersequence. In some embodiments, cleavage sites are two base pairsupstream of RpSpSp.

Exemplary oligonucleotide compositions are listed below.

SEQ ID Oligo Sequence Description Tm (° C.) NO: ONT-366dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGs All DNA 66.5 506dCsdTsdGsdCsdCsdAsdTsdA ONT-389 dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsS₇RSSRSSRS₅ 64.3 507 dCsdTsdGsdCsdCsdAsdTsdA ONT-390dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGs S₆RSSRSSRS₆ 64.6 508dCsdTsdGsdCsdCsdAsdTsdA ONT-391 dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsS₅RSSRSSRS₇ 64.3 509 dCsdTsdGsdCsdCsdAsdTsdA ONT-387rUrArUrGrGrCrArGrCrCrArGrGrCrArUrCrU complementary 510 rCrA RNA ONT-367dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGs All DNA 62.9 511dCsdTsdGsdCsdTsdCsdAsdC ONT-392 dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsS₇RSSRSSRS₅ 59.5 512 dCsdTsdGsdCsdTsdCsdAsdC ONT-393dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGs S₆RSSRSSRS₆ 60 513dCsdTsdGsdCsdTsdCsdAsdC ONT-394 dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsS₅RSSRSSRS₇ 59.5 514 dCsdTsdGsdCsdTsdCsdAsdC ONT-388rGrUrGrArGrCrArGrCrUrGrCrArArUrGrGrC complementary 515 rUrA RNA

EXAMPLE 7 Exemplary Chirally Controlled Oligonucleotide CompositionsProvide Higher Turn-over

In cases where the Tm of cleaved nucleic acid polymer fragments, forexample RNA fragments, to oligonucleotides is greater than aphysiological temperature, product dissociation may be inhibited andoligonucleotides may not be able to dissociate and find other targetstrands to form duplexes and cause the target strands to be cleaved. TheTm of ONT-316 (5-10-5 2′-MOE Gapmer) to complementary RNA is 76° C.After a cut or a few cuts in the RNA sequence complementary to theoligonucleotides, the 2′-MOE fragments may remain bound to RNA and thuscannot cause the other target molecules to be cleaved. Thermal meltingtemperatures of DNA strands generally are much lower when duplexed toRNA, for example, ONT-367 (63° C.) and ONT-392 60° C.). Additionally,thermal stability in DNA sequences is often relatively uniformlydistributed compared to 2′-MOE modified oligonucleotides. In someembodiments, oligonucleotides in provided chirally controlledoligonucleotide compositions do not contain 2′-modifications such as2′-MOE. In some embodiments, oligonucleotides in provided chirallycontrolled oligonucleotide compositions, which do not contain 2′-modifications such as 2′-MOE, more easily dissociate from nucleic acidpolymer cleavage fragments, and have higher turn-over thanoligonucleotides having 2′-modifications such as 2′-MOE. In someembodiments, the present invention provides an all DNA designs, in whicholigonucleotides do not have 2′-modifications. In some embodiments,chirally controlled oligonucleotide compositions whereinoligonucleotides having no 2′-modification provides higher turn-over ofa nuclease such as RNase H. In some embodiments, after cleavage RNase Hdissociates more easily from duplex formed by RNA and oligonucleotidesof provided chirally controlled oligonucleotide compositions. Usingsimilar protocols as described above, turn-over of two exemplarychirally controlled oligonucleotide compositions of oligonucleotide typeONT-367 and ONT-392 indeed showed higher turn-over rate than referencechirally uncontrolled oligonucleotide compositions (see FIG. 13).

EXAMPLE 8. Exemplary cleavage of FOXO1 mRNA

As exemplified in FIG. 14, controlled oligonucleotide compositions andmethods thereof in the present disclosure can provide controlledcleavage of nucleic acid polymers. In some embodiments, chirallycontrolled oligonucleotide compositions of the present inventionproduces altered cleavage pattern in terms of number of cleavage sites,location of cleavage sites, and/or relative cleavage percentage ofcleavage sites. In some embodiments, as exemplified by ONT-401 andONT-406, chirally controlled oligonucleotide compositions provide singlesite cleavage.

In some embodiments, only one component from RNA cleavage was detected.Without the intention to be limited by theory, Applicant notes that suchobservation could be due to the processive nature of RNase H enzymewhich could make multiple cuts on the same duplex resulting in muchshorter 5′—OH 3′—OH fragments.

Additional chirally controlled oligonucleotide compositions were furthertested. As described above, provided chirally controlled oligonucleotidecompositions provides unexpected results, for example, in terms ofcleavage rate and %RNA remaining in DNA/RNA duplex. See FIGS. 15-17.Exemple analytical data were presented in FIGS. 18-20. Without theintention to be limited by theory, Applicant notes that in someembodiments, cleavage may happen as depicted in FIG. 21. In FIG. 17, itis noted ONT-406 was observed to elicit cleavage of duplexed RNA at arate in slight excess of that of the natural DNA oligonucleotide ONT-415having the same base sequence and length. Applicant notes that chirallycontrolled oligonucleotide compositions of ONT-406, and other chirallycontrolled oligonucleotide compositions provided in this disclosure,have other preferred properties that an ONT-415 composition does nothave, for example, better stability profiles in vitro and/or in vivo.Additional exemplary data were presented in FIG. 25. Also, as will beappreciated by those skilled in the art, exemplary data illustrated inFIG. 26 and FIG. 27 confirm that provided exemplary chirally controlledoligonucleotide compositions, especially when so designed to control thecleavage patterns through patterns of backbone chiral centers, producedmuch better results than reference oligonucleotide compositions, e.g., astereorandom oligonucleotide composition. As exemplified in FIG. 26,controlled patterns of backbone chiral centers, among other things, canselectively increase and/or decrease cleavage at existing cleavage sitewhen a DNA oligonucleotide is used, or creates entirely new cleavagesites that do not exist when a DNA oligonucleotide is used (see FIG. 25,ONT-415). In some embodiments, cleavage sites from a DNA oligonucleotideindicate endogenous cleavage preference of RNase H. As confirmed by FIG.27, provided chirally controlled oligonucleotide compositions arecapable of modulating target cleavage rate. In some embodiments,approximately 75% of the variance in cellular activity is accounted forby differences in cleavage rate which can be controlled through patternsof backbone chiral centers. As provided in this Application, furtherstructural features such as base modifications and their patterns, sugarmodification and their patterns, internucleotidic linkage modificationsand their patterns, and/or any combinations thereof, can be combinedwith patterns of backbone chiral centers to provide desiredoligonucleotide properties.

EXAMPLE 9 Exemplary Allele-Specific Suppression of mHTT

In some embodiments, the present invention provides chirally controlledoligonucleotide compositions and methods thereof for allele-specificsuppression of transcripts from one particular allele with selectivityover the others. In some embodiments, the present invention providesallele-specific suppression of mHTT.

FIG. 22 illustrates exemplary chirally controlled oligonucleotidecompositions that specifically suppress transcripts from one allele butnot the others. Oligonucleotides 451 and 452 were tested withtranscripts from both exemplified alleles using biochemical assaysdescribed above. Allele-specific suppression is also tested in cells andanimal models using similar procedures as described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; U.S. patent application publication US2013/0197061; and Ostergaard et al., Nucleic Acids Research, 2013,41(21), 9634-9650. In all cases, transcripts from the target allele areselectively suppressed over those from the other alleles. As will beappreciated by those skilled in the art, exemplary data illustrated inFIG. 22 confirm that provided exemplary chirally controlledoligonucleotide compositions, especially when so designed to control thecleavage patterns through stereochemistry, produced much better resultsthan reference oligonucleotide compositions, in this case, astereorandom oligonucleotide composition. As confirmed by FIG. 22,patterns of backbone chiral centers can dramatically change cleavagepatterns (FIG. 22 C-E), and stereochemistry patterns can be employed toposition cleavage site at the mismatch site (FIG. 22 C-E), and/or candramatically improve selectivity between the mutant and wild type (FIG.22 G-H). In some embodiments, chirally controlled oligonucleotidecompositions are incubated with wtRNA and muRNA of a target and both theduplexes are incubated with RNase H.

Huntingtin Allele Tm

Mutant Huntingtin Allele ONT-453/ONT-451 38.8° C. Wild Type HuntingtinAllele ONT-454/ONT-451 37.3° C. Mutant Huntingtin Allele ONT-453/ONT-45238.8° C. Wild Type Huntingtin Allele ONT-454/ONT-452 36.5° C. MutantHuntingtin Allele ONT-453/ONT-450 40.3° C. Wild Type Huntingtin AlleleONT-454/ONT-450 38.8° C.

EXAMPLE 10 Exemplary Allele-Specific Suppression of FOXO1

In some embodiments, the present invention provides allele-specificsuppression of FOXO1.

FIG. 23 illustrates exemplary chirally controlled oligonucleotidecompositions that specifically suppress transcripts from one allele butnot the others. Oligonucleotides ONT-400, ONT-402 and ONT-406 weretested with transcripts from both exemplified alleles using biochemicalassays described above. Allele-specific suppression is also tested incells and animal models using similar procedures as described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; U.S. patent application publication US2013/0197061; Ostergaard et al., Nucleic Acids Research 2013, 41(21),9634-9650; and Jiang et al., Science 2013, 342, 111-114. Transcriptsfrom the target allele are selectively suppressed over those from theother alleles. In some cases, two RNAs with mismatch ONT-442 (A/G,position 7th) and ONT-443 (A/G, position 13th) from ONT-388 aresynthesized and are duplexed with ONT-396 to ONT-414. RNase H assay areperformed to obtain cleavage rates and cleavage maps.

EXAMPLE 11 Certain Exemplary Oligonucleotides and OligonucleotideCompositions

Stereorandom oligonucleotides with different 2′ substitution chemistriestargeting three distinct regions of FOXO1 mRNA with the thermal meltingtemperatures when duplexed with complementary RNA. The concentration ofeach strand was 1 uM in 1X PBS buffer.

SEQ ID Oligo Sequence Description Tm(° C.) NO: ONT-316TeosAeosGeos5mCeos5mCeosdAsdTsdTsdGs5 5-10-5 (2′-MOE 76.7 516mdCsdAsdGs5mdCsdTsdGs5mCeosTeos5mCe Gapmer) osAeos5mCeo ONT-355dTsdAsdGsdCsdCsdAsdTstsgscsasgscsdTsdGs 7-6-7 (DNA-2′-OMe- 71.2 517dCsdTsdCsdAsdC DNA) Gapmer ONT-361tsasgsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsdT 3-14-3 (2′-OMe- 65.8 518sdGsdCsdTscsascs DNA-2′-OMe) Gapmer ONT-367dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsd All DNA 62.9 519CsdTsdGsdCsdTsdCsdAsdC ONT-373 tsasgscscsdAsdTsdTsdGsdCsdAsdGsdCsdTsdG5-10-5 (2′-OMe 71.8 520 scstscsasc Gapmer) ONT-388rGrUrGrArGrCrArGrCrUrGrCrArArUrGrGrCr Complementary RNA 521 UrA ONT-302Teos5mCeos5mCeosAeosGeosdTsdTs5mdCs5 5-10-5 (2′-MOE 72.5 522mdCsdTsdTs5mdCsdAsdTsdTs5mCeosTeosGe Gapmer) os5mCeosAeo ONT-352dTsdCsdCsdAsdGsdTsdTscscststscsasdTsdTsd 7-6-7 (DNA-2′-OMe- 65.4 523CsdTsdGsdCsdA DNA) Gapmer ONT-358tscscsdAsdGsdTsdTsdCsdCsdTsdTsdCsdAsdTs 3-14-3 (2′-OMe- 62.6 524dTsdCsdTsgscsas DNA-2′-OMe) Gapmer ONT-364dTsdCsdCsdAsdGsdTsdTsdCsdCsdTsdTsdCsd All DNA 58.4 525AsdTsdTsdCsdTsdGsdCsdA ONT-370 tscscsasgsdTsdTsdCsdCsdTsdTsdCsdAsdTsdTs5-10-5 (2′-OMe 68 526 cstsgscsa Gapmer) ONT-386rUrGrCrArGrArArUrGrArArGrGrArArCrUrGr Complementary RNA 527 GrA ONT-315TeosGeosAeosGeosAeosdTsdGs5mdCs5mdCs 5-10-5 (2′-MOE 77.5 528dTsdGsdGs5mdCsdTsdGs5mCeos5mCeosAeos Gapmer) TeosAeo ONT-354dTsdGsdAsdGsdAsdTsdGscscstsgsgscsdTsdGs 7-6-7 (DNA-2′-OMe- 75.5 529dCsdCsdAsdTsdA DNA) Gapmer ONT-360tsgsasdGsdAsdTsdGsdCsdCsdTsdGsdGsdCsdT 3-14-3 (2′-OMe- 69 530sdGsdCsdCsastsas DNA-2′-OMe) Gapmer ONT-366dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsd All DNA 66.5 531CsdTsdGsdCsdCsdAsdTsdA ONT-372 tsgsasgsasdTsdGsdCsdCsdTsdGsdGsdCsdTsdG5-10-5 (2′-OMe 74.4 532 scscsastsa Gapmer) ONT-387rUrArUrGrGrCrArGrCrCrArGrGrCrArUrCrUr Complementary RNA 533 CrA

Additional exemplary stereorandom oligonucleotide compositions arelisted below.

SEQ ID Oligo Sequence (5′ to 3′) NO: ONT-41(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs 5345mCsAs5mCs5mC)_(MOE) ONT-70(Gs5mCs)_(MOE)d[GsTsTsTsGs5mCsTs5mCsTsTs5mCsTsTs](5mCsTsT 535sGs5mCGs)_(MOE)d[TsTsTsTs](TsT)_(MOE) ONT-83(GsTs5mCs5mCs5mCs)_(MOE)d(TsGsAsAsGsAsTsGsTs5mCs](AsAsTs 536Gs5mC)_(MOE) ONT-302(Ts5mCs5mCsAsGs)_(MOE)d[TsTs5mCs5mCsTsTs5mCsAsTsTs](5mCs 537TsGs5mCsA)_(MOE) ONT-315(TsGsAsGsAs)_(MOE)d[TsGs5mCs5mCsTsGsGs5mCsTsGs](5mCs5mCs 538AsTsA)_(MOE) ONT-316 (TsAsGs5mCs5mCs)_(MOE)d[AsTsTsGs5mCsAsGs5mCsTsGs5m]539 (CsTs5mCsAs5mC)_(MOE) ONT-352[TsCsCsAsGsTsTs](cscststscsas)_(OMe)d[TsTsCsTsGsCsA] 540 ONT-354[TsGsAsGsAsTsGs](CsCsTsGsGsCs)_(OMe)d[TsGsCsCsAsTsA] 541 ONT-355[TsAsGsCsCsAsTs](TsGsCsAsGsCs)_(OMe)d[TsGsCsTsCsAsC] 542 ONT-358(TsCsCs)_(OMe)d[AsGsTsTsCsCsTsTsCsAsTsTsCsTs](GsCsA)_(OMe) 543 ONT-360(TsGsAs)_(OMe)d[GsAsTsGsCsCsTsGsGsCsTsGsCsCs](AsTsA)_(OMe) 544 ONT-361(TsAsGs)_(OMe)d[CsCsAsTsTsGsCsAsGsCsTsGsCsTs](CsAsC)_(OMe) 545 ONT-364[TsCsCsAsGsTsTsCsCsTsTsCsAsTsTsCsTsGsCsA] 546 ONT-366[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA] 547 ONT-367[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 548 ONT-370(TsCsCsAsGs)_(OMe)d[TsTsCsCsTsTsCsAsTsTs](CsTsGsCsA)_(OMe) 549 ONT-372(TsGsAsGsAs)_(OMe)d[TsGsCsCsTsGsGsCsTsGs](CsCsAsTsA)_(OMe) 550 ONT-373(TsAsGsCsCs)_(OMe)d[AsTsTsGsCsAsGsCsTsGs](CsTsCsAsC)_(OMe) 551 ONT-440(UsAsGsCsCs)_(F)d[AsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 552 ONT-441(UsAsGsCsCs)_(F)d[AsTsTsGsCsAsGsCsTsGsC] 553 ONT-460(TsAsGsCsCs)_(OMe)d[AsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 554 ONT-450[AsTsTsAsAsTsAsAsAsTsTsGsTsCsAsTsCsAsCsC] 555

Exemplary RNA and DNA oligonucleotides are listed below.

SEQ Oli- ID go Sequence (5′ to 3′) NO: ONT-rGrGrUrGrCrGrArArGrCrArGrArCrUrGrArGrGrC 556 28 ONT-rUrGrCrArGrArArUrGrArArGrGrArArCrUrGrGrA 557 386 ONT-rUrArUrGrGrCrArGrCrCrArGrGrCrArUrCrUrCrA 558 387 ONT-rGrUrGrArGrCrArGrCrUrGrCrArArUrGrGrCrUrA 559 388 ONT-d[TAGCCATTGCAGCTGCTCAC] 560 415 ONT-rGrUrGrArGrCrGrGrCrUrGrCrArArUrGrGrCrUrA 561 442 ONT-rGrUrGrArGrCrArGrCrUrGrCrGrArUrGrGrCrUrA 562 443 ONT-rGrGrUrGrArUrGrArCrArArUrUrUrArUrUrArArU 563 453 ONT-rGrGrUrGrArUrGrGrCrArArUrUrUrArUrUrArArU 564 454

Exemplary chirally pure oligonucleotides are presented below. In someembodiments, the present invention provides corresponding chirallycontrolled oligonucleotide compositions of each of the followingexemplary oligonucleotides.

SEQ ID Oligo Stereochemistry/Sequence (5′ to 3′) Description NO: ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, 7S-(RSS)₃-3S 565 389Sp, Rp, Sp, Sp, Sp, Sp, Sp)- d[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, 6S-(RSS)₃-4S 566390 Rp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA] ONT-(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 5S-(RSS)₃-5S 567 391Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, 7S-(RSS)₃-3S 568392 Sp, Rp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, 6S-(RSS)₃-4S 569 393Rp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 5S-(RSS)₃-5S 570394 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S-1R 571 396Sp, Sp, Sp, Sp, Sp, Sp, Rp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 17S-RS 572 397Sp, Sp, Sp, Sp, Sp, Rp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 16S-(RSS) 573 398Sp, Sp, Sp, Sp, Rp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 15S-(RSS)-1S 574399 Sp, Sp, Sp, Rp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 14S-(RSS)-2S 575 400Sp, Sp, Rp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 13S-(RSS)-3S 576401 Sp, Rp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 12S-(RSS)-4S 577 402Rp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, 11S-(RSS)-5S 578403 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, 10S-(RSS)-6S 579 404Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, 9S-(RSS)-7S 580405 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, 8S-(RSS)-8S 581 406Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, 7S-(RSS)-9S 582407 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, 6S-(RSS)-10S 583 408Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, 5S-(RSS)-11S 584409 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 4S-(RSS)-12S 585 410Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 3S-(RSS)-13S 586411 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 2S-(RSS)-14S 587 412Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, S-(RSS)-15S 588413 Sp, Sp, Sp, Sp, Sp, Sp, Sp)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, (RSS)-16S 589 414Sp, Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- All-(Sp)- All S 590 421 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, 8S-(RSS)-3S- 591422 Sp, Sp, Rp, Sp, Sp, Sp, Sp)-C6-amino- (RSS)-2Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT- All-(Rp)- All R 592 455d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 13S-(RSS)-3S 593 451Sp, Rp, Sp, Sp, Sp, Sp, Sp)- d[AsTsTsAsAsTsAsAsAsTsTsGsTsCsAsTsCsAsCsC]ONT- (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 14S-(RSS)-2S 594452 Sp, Sp, Rp, Sp, Sp, Sp, Sp)-d[AsTsTsAsAsTsAsAsAsTsTsGsTsCsAsTsCsAsCsC] ONT- All-(Rp)- All R 595 75(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-(Sp, Rp, Rp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, SRRSR-11S-RSR596 76 Sp, Sp, Rp, Sp, Rp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 5R-10S-4R 59777 Sp, Rp, Rp, Rp, Rp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTs Ts5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT- All-(Sp)- All S 598 80(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTs Ts5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, 5S-10R-4S 59981 Rp, Sp, Sp, Sp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT- All-(Rp)- All R 600 82(GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs 5mCs](AsAsTsGs5mC)_(MOE)ONT- All-(Sp)- All S 601 84 (GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs5mCs](AsAsTsGs5mC)_(MOE) ONT-(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 5R-10S-4R 60285 Sp, Rp, Rp, Rp, Rp)- (GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs5mCs](AsAsTsGs5mC)_(MOE) ONT-(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, 5S-10R-4S 60386 Rp, Sp, Sp, Sp, Sp)- (GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs5mCs](AsAsTsGs5mC)_(MOE) ONT-(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 5R-2S-(RSS)₂-604 87 Rp, Rp, Rp, Rp, Rp)- 6R(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, 5S-(RRS)₃-5S605 88 Sp, Sp, Sp, Sp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTs Ts5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SR)₉S 606 89Sp, Rp, Sp, Rp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 7S-(RSS)₃-3S607 154 Sp, Sp, Sp, Sp, Sp)- d[Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC] ONT- All-(Rp)- All-R 608 75(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT- All-(Sp)- All-S 609 80(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE)

Additonal exemplary oligonucleotides targeting FOXO1 with Tm arepresented below. In some embodiments, the present invention providescorresponding chirally controlled oligonucleotide compositions of eachof the following exemplary oligonucleotides.

SEQ ID Oligo Sequence (5′ to 3′) Tm (° C.) NO: ONT-439[UsAsGs]_(F)d[CsCsAsTsTsGsCsAsGsCsTsGsCsTs][CsAsC]_(F) 68.3 610 ONT-440[UsAsGsCsCs]_(F)d[AsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 70.0 611 ONT-441[UsAsGsCsCs]_(F)d[AsTsTsGsCsAsGsCsTsGsC] 65.5 612 ONT-455 All-(Rp)- 66.8613 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-316[TsAsGs5mCs5mCs]_(MOE)d[AsTsTsGs5mCsAsGs5mCsTsGs][5 76.9 614mCsTs5mCsAs5mC]_(MOE) ONT-367 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]62.8 615 ONT-415 d[TAGCCATTGCAGCTGCTCAC] 72.6 616 ONT-416[TsAsGsCsCsAsTsTsGsCsAsGsCs]_(OMe)d[TsGsCsTsCsAsC] 78.4 617 ONT-421All-(Sp)- 59.2 618 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-394(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, 60.0 619Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-406(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, 58.5 620Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]

EXAMPLE 12 Exemplary Additional Controlled Cleavage by Provided ChirallyControlled Oligonucleotide Compositions

As will be appreciated by those skilled in the art, exemplary dataillustrated in FIG. 26 confirm that provided chirally controlledoligonucleotide compositions and methods thereof provided unexpectedresults compared to reference compositions, such as stereorandomoligonucleotide compositions. Among other things, chirally controlledoligonucleotide compositions can produce controlled cleavage patterns,including but not limited to controlling of positions of cleavage sites,numbers of cleavage sites, and relative cleavage percentage of cleavagesites. See also exemplary data presented in FIG. 27.

EXAMPLE 13 Stability of Chirally Controlled Oligonucleotide Compositions

As will be appreciated by those skilled in the art, exemplary dataillustrated in FIG. 26 confirm that stability of provided chirallycontrolled oligonucleotide compositions can be adjusted by varyingpatterns of backbone chiral centers. For exemplary data, see FIG. 7 andFIG. 28. An exemplary protocol for performing serum stability experimentis described below.

Protocol: P-stereochemically pure PS DNA (ONT-396-ONT-414 (single Rpwalk from 3′ end to 5′ end)), stereorandom PS DNA (ONT-367), all-Sp PSDNA (ONT-421) and all-Rp PS DNA (ONT-455) were incubated in Rat serum(Sigma, R9759) (0 h and 48 h) and analyzed by IEX-HPLC.

Incubation Method: 5 μL of 250 μM of each DNA solutions and 45 μL of Ratserum were mixed and incubated at 37° C. for each time points (0 h and48 h). At each time points, reaction was stopped by adding 25 μL of 150mM EDTA solution, 30 μL of Lysis buffer (erpicentre, MTC096H) and 3 μLof Proteinase K solution (20 mg/mL). The mixture was incubated at 60° C.for 20 min then 20 μL of the mixture was injected to IEX-HPLC andanalyzed.

Incubation Control Sample: Mixture of 5 μL of 250 μM of each DNAsolutions and 103 μL of 1× PBS buffer were prepared and 20 μL of themixture was analyzed by IEX-HPLC as controls in order to check theabsolute quantification.

Exemplary Analytical Method:

IEx-HPLC

A: 10 mM TrisHCl, 50%ACN (pH 8.0)

B: 10 mM TrisHCl, 800 mM NaCl, 50%ACN (pH 8.0)

C: Water-ACN (1:1, v/v)

Temp : 60° C.

Column: DIONEX DNAPac PA-100, 250×4 mm

Gradient:

Time Flow % A % B % C % D Curve 1 0.00 1.00 95.0 5.0 0.0 0.0 6 2 1.001.00 95.0 5.0 0.0 0.0 1 3 2.00 1.00 75.0 25.0 0.0 0.0 6 4 10.00 1.00 5.095.0 0.0 0.0 6 5 10.10 1.00 95.0 5.0 0.0 0.0 6 6 12.50 1.00 95.0 5.0 0.00.0 1Washing:

Time Flow % A % B % C % D Curve 1 0.01 1.00 0.0 0.0 100.0 0.0 6 2 5.501.00 0.0 0.0 100.0 0.0 1 3 5.60 1.00 0.0 100.0 0.0 0.0 6 4 7.50 1.00 0.0100.0 0.0 0.0 1 5 7.60 1.00 95.0 5.0 0.0 0.0 6 6 12.50 1.00 95.0 5.0 0.00.0 1Column Temperature: 60° C.Washing was performed every after the sample run.Percentage of remained PS DNA was calculated by the analysis of theratio from the 0 h to 48 h using the area of integration of HPLCchromatogram.

EXAMPLE 14 Exemplary Analytical Results (FIG. 19)

Peak assignments for FIG. 19 (Top panel, M12-Exp 11 B10, ONT-354, 30min)

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 2.341100.6 733.7 11.91 1390.6 1042.6 13.07 1500.08 1125.5 750.73 1805.291354.19 13.58 1603.39 1202.2 961.35 801.15 14.80 1589.9 1271.4 1059.518.59 1653.3 1323.3 1101.6

Assignment based on mass match Retention 3′-OH and time Observed5′-p-RNA 5′-OH, (minutes) MW fragment RNA DNA 2.34 2203.2  7 mer 11.914176 13 mer 13.07 4505.7 14 mer 5418.87 17 mer 13.58 4812.8 15 mer 14.806362.5 20 mer, ONT-387 18.59 6615.4 ONT-354

Peak assignments for FIG. 19 (Bottom panel, M12-Exp11 A10, ONT-315,30min)

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 4.011425.33 950.15 4.4 1100.83 733.69 4.94 1578.34 1051.54 6.21 1741.911161.89 870.37 1445.42 963.31 722.97 8.48 1610 1073.3 9.15 1391.2 1043.19.93 1763.4 1174.7 11.8 1602.3 1201.7 14.82 20.73 1809.94 1447.82 1205.9

Assignment based on mass match Retention 3′-OH and time Observed5′-p-RNA 5′-OH, (minutes) MW fragment RNA DNA 4.01 2853.45  9 mer 4.42203.66  7 mer 4.94 3158.47 10 mer 6.21 3487.52 11 mer 2892.84  9 mer8.48 3220.94 10 mer 9.15 4177 13 mer 9.93 3528.88 11 mer 11.8 4810 15mer 14.82 20 mer, ONT-387 20.73 7244.3 ONT-315

EXAMPLE 15 Exemplary Analytical Results (FIG. 30)

Peak assignments for FIG. 30 (Top panel, M12-Exp11 D2, ONT-367, 30min)

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 2.361120.28 746.25 3.15 1292.41 861.32 4.04 975.92 4.49 1140.6 759.78 5.831305.21 869.65 652.31 6.88 1923.23 1281.69 961.28 9.32 1390.76 1043.29833.72 9.96 1783.85 1187.98 891.6 712.94 11.01 1936.14 1289.93 1501.521125.4 899.89 11.93 1405.25 1053.78 842.84 13.15 1514.72 1135.72 14.811609.95 1287.53 1072.58 18.33 1587.9 1270.2 1058.3

Assignment based on mass match Retention 3′-OH and time Observed5′-p-RNA 5′-OH, (minutes) MW fragment RNA DNA 2.36 2242.56  7 mer 3.152586.82  8 mer 4.04 1953.84  6 mer 4.49 2283.2  7 mer 5.83 2612.42  8mer 6.88 3849.14 12 mer 9.32 4175.28 13 mer 9.96 3569.7 11 mer 11.013874.28 12 mer 4507.56 14 mer 11.93 4218.75 13 mer 13.15 4547.16 14 mer14.81 6441.8 20 mer, ONT-388 18.33 6355.6 ONT-367

Peak assignments for FIG. 30 (Bottom panel, M12-Exp21 NM Platel (pool)F11 ONT-406 30min

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 4.721140.6 759.78 9.46 1390.76 1043.29 833.72 16.45 1609.95 1287.53 1072.5819.48 1588.1 1270.4 1058.4

Assignment based on mass match Retention 3′-OH and time Observed5′-p-RNA 5′-OH, (minutes) MW fragment RNA DNA 4.72 2203.2 2283.2 7 mer9.46 4176 4175.28 13 mer 16.45 6362.5 6441.8 20 mer, ONT-388 19.486615.4 6355.9

Equivalents

Having described some illustrative embodiments of the invention, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the invention. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction.

Use of ordinal terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

The invention claimed is:
 1. A chirally controlled oligonucleotidecomposition comprising oligonucleotides of a particular oligonucleotidetype characterized by: 1) a common base sequence and length; 2) a commonpattern of backbone linkages; and 3) a common pattern of backbone chiralcenters, which composition is chirally controlled in that it isenriched, relative to a substantially racemic preparation ofoligonucleotides having the same base sequence and length, foroligonucleotides of the particular oligonucleotide type; wherein: theoligonucleotides of the particular oligonucleotide type each comprisethree or more chiral, modified phosphate linkages; the common pattern ofbackbone chiral centers comprises from 5′ to 3′ Rp(Sp)₂; the common basesequence has at least 17 bases; and the common pattern of backbonechiral centers comprises at least 50% of backbone chiral centers in theSp conformation.
 2. The composition of claim 1, wherein each chiral,modified phosphate linkage of the oligonucleotides of the particularoligonucleotide type independently has the structure of formula I:

wherein: P* is an asymmetric phosphorus atom and is either Rp or Sp; Wis O, S or Se; each of X, Y and Z is independently —O—, —S—, —N(—L—R¹)—,or L; L is a covalent bond or an optionally substituted, linear orbranched C1-C10 alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂ , —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)′, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—; R¹ is halogen, R,or an optionally substituted C₁-C₅₀ aliphatic wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, —Cy—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—; each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:two R′ on the same nitrogen are taken together with their interveningatoms to form an optionally substituted heterocyclic or heteroaryl ring,or two R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring; —Cy— is an optionally substituted bivalent ringselected from phenylene, carbocyclylene, arylene, heteroarylene, orheterocyclylene; each R is independently hydrogen, or an optionallysubstituted group selected from C₁-C₆ aliphatic, phenyl, carbocyclyl,aryl, heteroaryl, or heterocyclyl; and each —

— independently represents a connection to a nucleoside.
 3. Thecomposition of claim 2, wherein W is O, X is —S—, and Y and Z are —O—.4. The composition of claim 2, wherein X is —S— and —L—R¹ is nothydrogen.
 5. The composition of claim 2, wherein each chiral, modifiedphosphate linkage of the oligonucleotides of the particularoligonucleotide type is a phosphorothioate diester linkage.
 6. Thecomposition of claim 5, wherein the common pattern of backbone chiralcenters comprises from 5′ to 3′ SpSpRpSpSp.
 7. The composition of claim5, wherein the pattern of backbone chiral centers comprises from 5′ to3′ (Np)t(Rp)n(Sp)m, wherein t is 1, 2, 3, 4, 5, 6, 7 or 8, m is 2, 3, 4,5, 6, 7 or 8, n is 1, and each Np is independent Rp or Sp.
 8. Thecomposition of claim 7, wherein Np is Sp.
 9. The composition of claim 7,wherein t is greater than
 5. 10. The composition of claim 9, wherein thepattern of backbone chiral centers comprises (Sp)₂Rp(Sp)₂.
 11. Thecomposition of claim 5, wherein the oligonucleotides of the particularoligonucleotide type each comprise one or more phosphate diesterlinkages.
 12. The composition of claim 5, wherein the nucleobases of theoligonucleotides of the particular oligonucleotide type areindependently selected from adenine, thymine, cytosine, guanine, uraciland 5-methylcytosine.
 13. The composition of claim 5, wherein theoligonucleotides of the particular oligonucleotide type each comprise amodified sugar.
 14. The composition of claim 13, wherein the modifiedsugar comprises a 2′-modification.
 15. The composition of claim 14,wherein the 2′-modification is 2′ —OR¹, wherein R¹ is optionallysubstituted C₁-C₆ aliphatic.
 16. The composition of claim 14, whereinthe 2′-modification is 2′ —OMe.
 17. The composition of claim 14, whereinthe 2′-modification is 2′ —OCH₂CH₂OMe.
 18. The composition of claim 5,wherein the oligonucleotides of the particular oligonucleotide type eachcomprise a locked nucleic acid sugar.
 19. The composition of claim 13,wherein the modified sugar comprises a bivalent substituent —L—, wherein—L— is —O—CH₂— between C₂ and C₄ of the sugar, wherein the —CH₂— isoptionally substituted.
 20. The composition of claim 13, wherein themodified sugar comprises a bivalent substituent —L—, wherein —L— is—O—CH₂(Et)— between C₂ and C₄ of the sugar.
 21. The composition of claim1, wherein the oligonucleotides of the particular oligonucleotide typeeach comprise at least one chiral, modified phosphate linkage having thestructure of formula I:

wherein: P* is an asymmetric phosphorus atom and is either Rp or Sp; Wis O, S or Se; each of X, Y and Z is independently —O—, —S—, —N(—L—R¹),or L; L is a covalent bond or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂ —, —Cy—, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—; R¹ is halogen, R, oran optionally substituted C₁-C₅₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, —Cy—,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—; each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or: twoR′ on the same nitrogen are taken together with their intervening atomsto form an optionally substituted heterocyclic or heteroaryl ring, ortwo R′ on the same carbon are taken together with their interveningatoms to form an optionally substituted aryl, carbocyclic, heterocyclic,or heteroaryl ring; —Cy— is an optionally substituted bivalent ringselected from phenylene, carbocyclylene, arylene, heteroarylene, orheterocyclylene; each R is independently hydrogen, or an optionallysubstituted group selected from C₁-C₆ aliphatic, phenyl, carbocyclyl,aryl, heteroaryl, or heterocyclyl; and each —

— independently represents a connection to a nucleoside.
 22. Thecomposition of claim 21, wherein the chiral, modified phosphate linkagehaving the structure of formula I is a phosphorothioate diester linkage.23. The composition of claim 22, wherein the oligonucleotides of theparticular oligonucleotide type each comprise a modified sugar.
 24. Thecomposition of claim 23, wherein the modified sugar comprises a2′-modification.
 25. The composition of claim 24, wherein the2′-modification is 2′ —OR¹, wherein R¹ is optionally substituted C₁-C₆aliphatic.
 26. The composition of claim 25, wherein the 2′-modificationis 2′ —OMe.
 27. The composition of claim 25, wherein the 2′-modificationis 2′ —OCH₂CH₂OMe.
 28. The composition of claim 23, wherein the modifiedsugar is a locked nucleic acid sugar.
 29. The composition of claim 23,wherein the modified sugar comprises a bivalent substituent —L—, wherein—L— is —O—CH₂— between C₂ and C₄ of the sugar, wherein the —CH₂ —isoptionally substituted.
 30. The composition of claim 23, wherein themodified sugar comprises a bivalent substituent —L—, wherein —L— is—O—CH₂(Et)— between C₂ and C₄ of the sugar.
 31. The composition of claim5, wherein at least about 20% of the oligonucleotides in the compositionhave the common base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.
 32. Thecomposition of claim 5, wherein the common base sequence has at least 19bases.
 33. The composition of claim 5, wherein the common base sequencehas at least 20 bases.
 34. The composition of claim 8, wherein m is 3,4, 5, 6, 7 or
 8. 35. The composition of claim 8, wherein t is 6, 7 or 8.